Compositions and Methods for Improved Skin Care

ABSTRACT

Compositions and methods for administering collagen to a human subject have been developed. The collagen-containing lipid vesicles of the invention provide a delivery system for human collagen which eliminates problems associated with chemical and physical instability of the collagen as well as immune responses to non-human collagen.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a continuation application of co-pending applicationSer. No. 11/542,554, filed Oct. 3, 2006; which claims the benefit ofprovisional patent applications Ser. No. 60/723,043, filed Oct. 3, 2005;and Ser. No. 60/833,045, filed Jul. 25, 2006, which are herebyincorporated by reference in their entirety, including any figures,tables, or drawings.

BACKGROUND OF THE INVENTION

The skin is the largest organ in the human body and consists essentiallyof two primary layers—the epidermis and the dermis. The epidermis is theoutermost layer and, among other things, controls water loss from cellsand tissue. The dermis is the layer below the epidermis and containsblood vessels, lymph vessels, hair follicles and sweat glands. Below thedermis is the hypodermis. Although the hypodermis is considered to bepart of the integumentary system, it is not generally considered to be alayer of the skin. The hypodermis is used mainly for fat storage.

The outermost epidermis is made up of stratified squamous epitheliumwith an underlying basement membrane. It contains no blood vessels, andis nourished by diffusion from the dermis. The main type of cells thatmake up the epidermis are keratinocytes, with melanocytes and Langerhanscells also present. The epidermis can be further subdivided into thefollowing strata (beginning with the outermost layer): corneum, lucidum,granulosum, spinosum, basale. Cells are formed through mitosis at theinnermost layers. They move up the strata changing shape and compositionas they differentiate and become filled with keratin. They eventuallyreach the corneum and become sloughed off. This process is calledkeratinization and takes place within about 30 days.

The dermis consists largely of the protein collagen, which forms anetwork of cross-linked fibers providing a framework for blood vesselsand cell growth. Because it is the primary component of the dermis,collagen acts as the support structure for the skin. The health andstability of collagen is a critical factor in determining the contour,wrinkles and lines in the skin.

Hyaluronic acid (HA) is a natural substance found in all livingorganisms in soft connective tissues, the vitreous humor of the eye,some cartilage and joint fluids, and skin tissue. In skin tissue,hyaluronic acid is a jelly like substance that fills the space betweencollagen and elastin fibers. Its role is to provide a mechanism oftransport of essential nutrients from the bloodstream to living skincells, to hydrate the skin by holding in water, and to act as acushioning and lubricating agent against mechanical and chemical damage.Over time however, due to aging and other external factors, the body'snatural supply of hyaluronic acid is slowly absorbed and disappearsgradually.

There are 7 to 8 grams of hyaluronic acid in adults, 50% of which(3.5-4.0 g) are found in the skin. It is distributed at approximately0.5 mg/g in the dermis and about 0.1 mg/g in the epidermis. It is notspecific to species or organs and is thought to be without risk ofpromoting allergy or causing a foreign body reaction.

Administration of hyaluronic acid can be used to help hydrate the skin,smooth wrinkles, and generally improve skin appearance. Theeffectiveness of such administration is limited by the relatively rapidbreakdown of this compound caused by enzymes that exist naturally in thebody. In recent years, efforts have been made to create longer lastinghyaluronic acid compositions. Specifically, cross-linked hyaluronic acidcompounds have now been developed that significantly increase thehalf-life of HA in the body. These compositions, which have reducedwater solubility, are injected for cosmetic treatment. These injectionsare similar to collagen injections and, apart from being non-allergenic,have the same limitations and drawbacks. These limitations and drawbacksare discussed in more detail below.

Collagen, a naturally occurring fibrous protein found in both humans andanimals, provides structural support for bones, tendons, ligaments, andblood vessels, in addition to its role in the skin. Collagen is the mostabundant protein in the body.

There are several major types of collagen, which give rise to thevariety of structural and functional properties that collagen exhibitsthroughout the body. With age or injury, the collagen in a person beginsto weaken and lose its elasticity. In the skin, this process eventuallyresults in the appearance of wrinkles.

The basic structural unit of a collagen fiber is tropocollagen. Itconsists of a triple helix of three intertwined peptide chains ofapproximately 1000 amino acid residues. The basic polypeptide unit ofthe peptide chain is a repeating sequence of 3 amino acids, where everythird residue is a glycine, and the other two alternate between prolineand hydroproline. It is important to the stabilizing feature of thecollagen fiber that the glycine residue is every third residue becauseits small side chain allows for tight coiling of the three helices,providing a strong stabilizing structure.

In young skin, the collagen remains intact and elastic, however, as theskin ages, the support structure weakens, the skin loses elasticity andthe collagen support wears down from the cumulative stress of, forexample, facial expressions. This causes lines and wrinkles to appear inthe skin.

Collagen replacement therapy can be used to treat conditions associatedwith the breakdown or loss of collagen. For example, skin wrinkles canbe treated by injecting highly purified collagen into the dermis.Injection of collagen has also been used to soften scar tissue andcreate fuller lips.

Current collagen replacement therapies include collagen injections inwhich purified animal collagen is used to replace lost tissue. Zyderm®and Zyplast® are bovine collagen implants that are injected into thedermis. There they become incorporated into the human collagen frameworkand replenish the skin's natural collagen thereby restoring the supportstructure and the contour of the skin. This injection therapy enhancesand improves the natural appearance of skin and smoothes facial linesand scars.

Procedures involving injecting collagen are not without risk. Forexample, bovine collagen injections can cause allergic reactions such asredness, swelling, firmness, itching and, in rare instances, abscessformation. Worse, some physicians have reported the occurrence ofconnective tissue diseases such as rheumatoid arthritis, systemic lupuserythematosus, dermatomyositis (DM), and polymyositis (PM) subsequent tocollagen injections in patients with no previous history of thesedisorders.

Also, the injection process itself poses certain challenges. Forexample, the practitioner injecting collagen (and/or hyaluronic acid)must control the depth, orientation and position of the needle at aparticular injection site, while providing an inward force on theplunger that is sufficient to force a controlled flow rate of highviscosity collagen out of the needle and into the exact location in thedermis that will provide the desired cosmetic effect. The locating ofthe needle tip at the proper depth within the dermis is also difficultfor the practitioner. To engage the tip of the needle at the properinjection depth, the practitioner may move the needle inwardly andoutwardly with respect to the surface of the skin (epidermis). However,there is no visual reference point, other than the end of the syringebody, from which the practitioner can easily determine the extent thatthe needle extends into the dermis. Thus, the needle tip may be placedtoo deep, or too shallow, for the intended application. It should beappreciated that the person (practitioner) injecting the collagen musthave good, steady control of the fingers, hand and arm and also haveexcellent eye-hand coordination to be an effective provider of cosmeticcollagen injections. These qualities are not always present inindividuals, and this has limited the availability of collagen therapyto patients.

In addition to injection, collagen may be delivered to the skin bytopical application. Unfortunately, such topical collagen therapy hasproven less than effective as conventional forms of collagen do notappear to penetrate into the dermis. As noted above, the skin consistsof multiple layers and is extremely complex in terms of its function aswell as its chemical make-up. Transdermal (through the skin) applicationof medicines and other substances poses a wide range of formulationhurdles. The ability to deliver desired substances to a layer within theskin is equally, if not even more, difficult.

Various means for delivery of substances to or into the skin have beenproposed. U.S. Pat. No. 5,354,564 discloses personal care productscomprising an aqueous dispersion of particles of silicone wherein saidparticles have a surface modifier adsorbed on the surface thereof in anamount sufficient to achieve a particle size of less than about 400nanometers (nm).

U.S. Pat. No. 5,660,839 discloses incorporating deformable hollowparticles into cosmetic and/or dermatological compositions containingfatty substances, for markedly reduce or eliminate the sticky and/orgreasy feel attributed to these fatty substances.

U.S. Pat. No. 5,667,800 discloses an aqueous suspension of solid lipoidnanoparticles, comprising at least one lipid and preferably also atleast one emulsifier, for topical application to the body.

U.S. Pat. No. 5,780,060 discloses microcapsules with a wall ofcrosslinked plant polyphenols and compositions containing them. Themicrocapsules are obtained by the interfacial crosslinking of plantpolyphenols, particularly flavonoids.

U.S. Pat. Nos. 5,851,517 and 5,945,095 disclose compositions including adispersion of polymer particles in a non-aqueous medium. A dispersion ofsurface-stabilized polymer particles can be used in a non-aqueousmedium, in a cosmetic, hygiene or pharmaceutical composition. Thedispersions may, in particular, be in the form of nano-particles ofpolymers in stable dispersion in a non-aqueous medium.

U.S. Pat. Nos. 5,759,526 and 5,919,487 disclose nanoparticles coatedwith a lamellar phase based on silicone surfactant and compositionscontaining them. The nanoparticles, and in particular nanocapsules,provided with a lamellar coating obtained from a silicone surfactant,can be used in a composition, in particular a topical composition, fortreatment of the skin, mucosae, nails, scalp and/or hair.

U.S. Pat. No. 5,188,837 discloses a microsuspension system and methodfor its preparation. The microsuspension contains lipospheres which aresolid, water-insoluble microparticles that have a layer of aphospholipid embedded on their surface. The core of the liposphere is asolid substance to be delivered or a substance to be delivered that isdispersed in an inert solid vehicle such as a wax.

U.S. Pat. No. 4,919,841 discloses a process for preparing encapsulatedactive particles by the steps of: dispersing active materials in moltenwax; emulsifying the active/wax dispersion in an aqueous surfactantsolution for no longer than 4 minutes; quenching the capsules bycooling; and retrieving solidified capsules. Examples of activematerials are fragrances.

Each of these methods has disadvantages, particularly with respect tothe delivery of collagen and/or hyaluronic acid.

Liposomes are vesicular lipid membrane structures that enclose, forexample, a volume of water. The existence of liposomes has been knownfor many years. In the early 1900's, researchers, studying isolatedlecithin (phosphatidylcholine), cephalin(phosphatidylethanolamine/phosphatidylserine), phrenosin (galactosylceramide) and kerasin (glucosyl ceramide), found that all of thesemolecules would swell in water to form hydrated multilamellar layers,consisting of lipid bilayers separated by water. Also, mixtures of ionicand nonionic lipids dispersed in water were found to form stable“emulsions” in which the lipid molecules take up positions side by sideto form a homogeneous mixed phase. These emulsions were the equivalentsof what are now called multilamellar liposomes.

Physical and chemical studies have shown that amphiphiles form certainpreferred arrays in the presence of water. Formation of these arrays,which include micelles, monolayers and bimolecular layers, is driven bythe need for the polar head groups, which may be ionogenic or not, toassociate with water and the need of the apolar, hydrophobic tail to beexcluded from water. Exactly which type of structure is assumed dependsupon the nature of the amphiphile, its concentration, the presence ofother amphiphiles, temperature, and presence of salt and other solutesin the aqueous phase.

Until recently, liposome technology has been concerned mostly withvesicles composed of phospholipids, predominantly phosphatidylcholine,and these continue to be the focus of most publications and patents.However, although phospholipids are suitable for certain pharmaceuticalapplications, phospholipid liposome technology has been beset by seriousproblems, for example, phospholipids turn over rapidly in vivo and areunstable in storage. Also, they are labile and expensive to purify orsynthesize, and the manufacture of phospholipid liposomes is difficultand costly to scale up.

Although liposomes are well known in the art, there are no previousreports of their use to efficiently deliver collagen and/or hyaluronicacid in a skin care formulation.

BRIEF SUMMARY OF THE INVENTION

The subject invention pertains to new and advantageous skin carecompositions. In a preferred embodiment, the subject invention provideslipid vesicles (liposomes) incorporating hyaluronic acid. Particularlypreferred is the use of cross-linked hyaluronic acid. Free, ornon-crosslinked hyaluronic acid may also be used as a component of thecomposition. In a further preferred embodiment, the compositions of thesubject invention also comprise vesicles containing collagen. Thecompositions may also contain additional skin care agents.

The subject invention further pertains to methods of using such lipidvesicles for delivery of hyaluronic acid, collagen, and other activeingredients to a patient to achieve enhanced skin care.

Hyaluronic acid is a naturally occurring sugar that exists in all livingorganisms and is a universal component of the extra-cellular spaces ofbody tissues. It functions by holding together collagen and elastin,thus providing a framework for the skin. When applied to the skinaccording to the subject invention, preferably in cross-linked gel form,hyaluronic acid acts as a dermal filler by binding to water andproviding volume to easily fill in facial lines and cause visibleplumping of the skin. When used according to the subject inventionhyaluronic acid acts as an efficient hydrating agent.

In one embodiment, the present invention provides a skin carecomposition with cross-linked hyaluronic acid that further comprises asafe and effective amount of collagen, wherein the formulationfacilitates the active ingredients passing through the epidermis andthus being released within the dermis of the skin. In a furtherembodiment, the human collagen and/or crosslinked HA may be delivered tothe epidermis as well. Accordingly, the present invention is useful inregulating and/or improving the condition of the skin (including theappearance and/or feel of the skin) by efficiently delivering hyaluronicacid and/or collagen to the appropriate location within the skin.

In addition, the use of human collagen (e.g., recombinant human collagenor human collagen isolated from human tissue or cultured humanfibroblasts), or a fragment thereof, is advantageous for avoidingundesired side effects such as allergic or autoimmune reactions.Advantageously, hyaluronic acid does not present a significant risk ofan allergic reaction.

The present invention also relates to methods of using such compositionsto regulate and/or improve the condition of skin. The methods of thesubject invention generally include the step of topically applying thecompositions to the skin (epidermis) of the patient needing suchtreatment, wherein a therapeutically effective amount of suchcomposition is applied. Advantageously, the present invention providescompositions and methods for combating the aging of skin, whereincombating the aging of skin can include, for example, hydration of theskin, treating the appearance of wrinkles, fine lines, and other formsof undesirable skin texture. By presenting collagen and/or hyaluronicacid into the dermal and/or epidermal layer(s) of the skin, the form,strength, as well as function of the skin is enhanced.

In certain embodiments, the compositions of the subject inventioncomprise a dispersion of lipid vesicles that contain agents, in additionto hyaluronic acid and collagen, that are useful in delaying,minimizing, or eliminating skin aging, wrinkling, and/or otherhistological changes typically associated with the intrinsic conditions(such as aging, menopause, acne, etc.) and extrinsic conditions (such asenvironmental pollution, wind, heat, low humidity, harsh surfactants,etc.).

In an exemplary embodiment of the invention non-phospholipidpaucilamellar lipid vesicles incorporating human collagen and hyaluronicacid are used to deliver collagen to the skin of a human subject.Non-phospholipid paucilamellar lipid vesicles are particularlyadvantageous for use in the invention as such vesicles are stable andinexpensive to manufacture, and also feature a large cavity size forholding collagen. In an alternative embodiment, cyclodextrins are usedto deliver the active agents to the dermis layer of the skin.

DETAILED DESCRIPTION

The present invention is directed to materials and methods for thetopical administration of a therapeutically effective amount ofhyaluronic acid and/or collagen to a specific layer within the skin inorder to improve the condition of the skin. Accordingly, in a preferredembodiment, the present invention provides compositions, and methods forusing such compositions, comprising a dispersion of lipid vesicles thatcontain at least hyaluronic acid and/or collagen (and, optionally, otherskin care agents), wherein the lipid vesicles facilitate penetrationthrough the epidermis and dispersal of the vesicle contents, into thedermis layer of the skin.

Improvement of skin condition is often desired due to conditions thatmay be induced or caused by factors internal and/or external to thebody. Examples include, but are not limited to, environmental damage,smoking, radiation exposure (including ultraviolet radiation),chronological aging, menopausal status (e.g., post-menopausal changes inskin), stress, diseases, etc.

The present invention is useful for therapeutically and/orprophylactically improving visible and/or tactile characteristics inskin. For example, in one embodiment, the length, depth, and/or otherdimension of lines and/or wrinkles are decreased and hydration isachieved.

“Improving skin condition” includes prophylactically preventing ortherapeutically treating a skin condition, and may involve one or moreof the following benefits: thickening of skin, preventing loss of skinelasticity, and a reduction in lines or wrinkles.

Following are additional definitions relevant to the subject invention.It should be appreciated that the following definitions are usedthroughout this application. Unless otherwise defined, all technicalterms used herein have the same meaning as commonly understood by one orordinary skill in the art to which this invention belongs.

The term “epidermis” or “epidermal,” as used herein, refers to theoutermost layer of the skin.

The term “topical application,” as used herein, means to apply or spreadthe compositions of the present invention onto the surface of theepidermis tissue.

The term “dermatologically-acceptable,” as used herein, means that thecompositions or components thereof so described are suitable for use incontact with mammalian epidermal tissue without undue toxicity,incompatibility, instability, allergic response, and the like.

The term “therapeutically effective amount,” as used herein, refers toan amount of a compound (such as collagen) or composition sufficient toinduce a positive benefit, preferably a positive skin appearance and/orfeel. In accordance with the subject invention, the therapeuticallyeffective amount is an amount of collagen, either alone or incombination with other agents, that regulates and/or improves the skin,but where the amount is low enough to avoid serious side effects, i.e.,to provide a reasonable benefit to risk ratio, within the scope of soundjudgment of the skilled artisan.

The term “sagging” as used herein means the laxity, slackness, or thelike condition of skin that occurs as a result of loss of, damage to,alterations to, and/or abnormalities in dermal structure and/orfunction.

The terms “smoothing” and “softening,” as used herein, refer to alteringthe surface of the epidermis tissue such that its tactile feel isimproved.

“Signs of skin aging” include, but are not limited to, all outwardvisibly and tactilely perceptible manifestations as well as any othermacro or micro effects due to skin aging. Such signs may be induced orcaused by intrinsic factors or extrinsic factors, e.g., chronologicalaging and/or environmental damage. These signs may result from processeswhich include, but are not limited to, the development of texturaldiscontinuities such as wrinkles and coarse deep wrinkles, skin lines,crevices, bumps, large pores (e.g., associated with adnexal structuressuch as sweat gland ducts, sebaceous glands, or hair follicles), orunevenness or roughness, loss of skin elasticity, sagging (includingpuffiness in the eye area and jowls), loss of skin firmness, loss ofskin tightness, loss of skin recoil from deformation, discoloration(including undereye circles), blotching, sallowness, hyperpigmented skinregions such as age spots and freckles, keratoses, abnormaldifferentiation, hyperkeratinization, elastosis, collagen breakdown, andother histological changes in the stratum corneum, dermis, epidermis,the skin vascular system (e.g., telangiectasia or spider vessels), andunderlying tissues, especially those proximate to the skin.

As used herein, “shear mixing” means the mixing of a lipophilic phasewith an aqueous phase under turbulent or shear conditions that provideadequate mixing to hydrate the lipid and form lipid vesicles

By the terms “disperse” and “dispersion” are meant dissolution orforming a suspension or colloid to yield a flowable phase.

As used herein, a “nucleic acid” or a “nucleic acid molecule” means achain of two or more nucleotides such as RNA (ribonucleic acid) and DNA(deoxyribonucleic acid). A “recombinant” nucleic acid molecule is onemade by an artificial combination of two otherwise separated segments ofsequence, e.g., by chemical synthesis or by the manipulation of isolatedsegments of nucleic acids by genetic engineering techniques.

The terms “protein” and “polypeptide” are used synonymously to mean anypeptide-linked chain of amino acids, regardless of length orpost-translational modification, e.g., glycosylation or phosphorylation.A “purified” polypeptide is one that has been substantially separated orisolated away from other polypeptides in a cell or organism in which thepolypeptide naturally occurs (e.g., 90, 95, 98, 99, 100% free ofcontaminants).

When referring to a nucleic acid or polypeptide, the term “native”refers to a naturally-occurring nucleic acid or polypeptide.

The compositions of the present invention, which enable dermal layerdispersion of collagen, are useful for improving the skin, includingimproving skin appearance and/or feel. For example, compositions of thepresent invention are useful for improving the appearance of skincondition by providing a visual improvement in skin appearance followingapplication of the composition to the skin.

Advantageously, the compositions of the present invention may haveadditional desirable properties, including stability, long shelf-life,absence of significant skin irritation, and good aesthetics. In certainembodiments, to accomplish such additional benefits, the compositions ofthe invention further comprise agents, in addition to the collagen, thatpromote composition stability, reduce skin irritation, and/or enhancethe aesthetic appeal of the composition.

Examples of good aesthetics include compositions, such as luxuriouscreams and lotions, that (i) are light and nongreasy, (ii) have asmooth, silky feel upon the skin, (iii) spread easily, and/or (iv)absorb quickly. Other examples of good aesthetics include compositionsthat have a consumer acceptable appearance (i.e. no unpleasant odor ordiscoloration present), and provide good skin feel.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In the case of conflict,the present specification, including definitions, will control. Inaddition, the particular embodiments discussed below are illustrativeonly and not intended to be limiting.

Hyaluronic Acid

Hyaluronic acid (HA) is present throughout nature and is a combinationof repeated disaccharide units of glucuronic acid and N-acetylglucosamine. It is polyionic and has an axial hydrophobic part and acentral hydrophilic part. Hyaluronic acid is very sensitive tohyaluronidases and, therefore, has a short half-life in the body. Inorder to make it a wrinkle-filling product that has sufficientsustainability over time it can be cross-linked.

Numerous substances can be used to cross-link hyaluronic acid includingformaldehyde, epoxides, polyaziridyl compounds, divinyl sulfone andothers. One cross-linking agent is divinyl sulfone. This substancereacts readily with hyaluronic acid in aqueous alkaline solutions,thereby providing cross-linked HA gels. These gels swell in water. Theswelling ratio depends upon the degree of cross-linking of the gel. Thedegree of cross-linking can be controlled by changing several factorsincluding the molecular weight of the HA, its concentration in thereaction mixture, the alkali concentration and the polymer/DVS ratio.The swelling ratio of these gels can be from 20 up to 8000, and more,depending upon the reaction parameters.

An even more preferred cross-linking agent is 1,4-butanediol diglycidylether (BDDE).

The swelling ratio of cross-linked HA gels is substantially greater thanthe swelling ratio of cross-linked gels of other polysaccharidesobtained under the same reaction conditions. This can probably beexplained by the unique nature of HA (as compared to otherpolysaccharides) and its water solutions. In water, a large molecule ofHA forms a very flexible, long random coil that takes up a large volumein the solution.

The unique property of HA to give highly swollen cross-linked gels canbe used to effect modification of the properties of cross-linked gelsmade of mixtures of HA with other hydrophilic polymers. These polymersinclude other polysaccharides, synthetic and natural, such ashydroxyethyl cellulose, carboxymethyl cellulose, xanthan gum,glycosaminoglycans, proteins and glyco proteins of various types, suchas collagen, elastin, albumin, globulin, etc, sulfated proteins,synthetic water-soluble polymers, such as polyvinyl alcohol and itsco-polymers, co-polymers of poly-(hydroxyethyl)methacrylate and thelike. Any polymer soluble in water or aqueous alkaline solutions andcontaining groups capable of reacting with DVS, namely, hydroxyl, aminoor sulfhydryl groups, can be used to obtain highly swollen cross-linkedmixed gels of HA.

Another convenient method of obtaining cross-linked hyaluronic acid ormixed hyaluronic acid and other polymer gels comprises treating drypolymer preparations, i.e., in the form of a film with a cross-linkingagent and subsequent swelling of the product in the desired medium.

In one embodiment, linear HA of less than 500,000 Da can be used.

As described herein, the administration of HA can be used toadvantageously improve the condition of the skin. Also, because of itsmolecular structure, HA can be used to entrap and deliver additionalactive agents (drugs). There are several methods for combining a drugwith the HA gel and, accordingly, several types of products that can beobtained. One of the methods comprises diffusing a drug into an HA gelwhen the gel is put into a solution of the drug. The product obtained bythis method is a gel in which a drug substance is uniformly dispersed.

The same type of product can be obtained by dehydrating a hyaluronicacid gel and reswelling it in a drug solution. To dehydrate a gel onecan use a water-miscible organic solvent or alternatively, water from agel can be removed by drying. Preferable solvents are ethanol andisopropanol, and ketones such as acetone, though other solvents can alsobe used.

Yet another method can be used to obtain products of this type. Thismethod comprises allowing a concentrated hyaluronic acid gel resultingfrom a cross-linking reaction previously carried out in a relativelyconcentrated solution of hyaluronic acid to swell in a solution of adrug substance.

Although these three methods all result in products that are essentiallythe same, each of the methods has certain advantages when compared toany of the other methods for any specific product and, hence, the choiceof method should be made with consideration given to such parameters asnature of the drug, the desired concentration of the drug in the system,the delivery rate, etc.

A drug delivery system of another type according to the presentinvention is one in which a drug is covalently attached tomacromolecules of hyaluronic acid and/or other polymers forming a gel.These systems are characterized by a substantially slower rate ofdelivery than those described above. Delivery of a drug from thesesystems occurs when the gel is degraded in the living body. Thedegradation process is usually slower than diffusion. The rate of thedegradation process can be controlled by several means, includingadjusting the density of the cross-links in the gel or byco-cross-linking hyaluronic acid with polymers which can be degraded inthe body more easily than hyaluronic acid, e.g., proteins. By changingthe concentration of such polymer components in the mixed gels, one canconveniently control their rate of degradation and, thus, the rate ofdrug delivery.

Another possibility of drug delivery for this type of product involvesthe use of such chemical bonds for attachment of a drug to polymericmolecules forming a gel that has a controllable rate of hydrolysis in aphysiological environment.

To obtain this type of a product one can use a drug substance that canreact with a cross-linking agent. Yet another method can be used toobtain this product. This method comprises chemically modifying across-linked gel after its formation, using the reactive hydroxyl groupsof hyaluronic acid or the reactive groups of the polymersco-cross-linked with the hyaluronic acid to which a drug substance canbe attached by numerous chemical reactions. Alternatively, additionalreactive groups can be introduced by chemical treatment of across-linked gel which affects the macromolecules of hyaluronic acid orco-cross-linked polymers and a drug can be covalently attached to thesenewly formed reactive groups. The active agents can be cosmetic,dermatological, and pharmaceutical active agents.

Suitable active agents include, but are not limited to, ceramides;vitamins; antioxidants; free radical scavengers; moisturizing agents;anti-seborrhoeic agents; anti-UV agents; keratolytic agents;anti-inflammatory agents; melanoregulators; liporegulators; anti-ageingagents; antibacterial agents; agents for combating hair loss; vascularprotectors; anti fungal agents; skin conditioners; immunomodulators;nutrients and essential oils; retinoids; anesthetics; preservatives;antiseptics; emollients; lubricants; humectants; pigments; dyes; hydroxyacids, such as, alpha hydroxy acids, and beta hydroxy acids; elastin;hydrolysates; epidermal growth factor; soybean saponins; andmucopolysaccharides.

Several forms of hyaluronic acid have been developed by cross-linkingthe acid with other natural acids or chemical compounds to form gelsthat improve skin condition.

At present several commercial preparations are available. These includeHylaforre, extracted from coxcomb and marketed by the Genzyme Companyand Restylane® which is produced by bacteria fermentation process usingStreptococcus equi spices and crosslinked with BDDE. These twocompositions have the common feature of being biphasic injectablesubstances, with particles of hyaluronic acid cross-linked to a greateror lesser degree and suspended in a more fluid or even non-cross-linkedpreparation.

Monophasic gel preparations of hyaluronic acid are contained in otherproducts such as Juvéderm®, Hydra Fillé and Esthélis®. As with theRestylane® each of these is produced with BDDE, although the process isdifferent resulting in the monophasic gel.

Tn one embodiment, the crosslinked hyaluronic acid is produced as amonophasic composition. After linearising the spine of the nativehyaluronic acid, cross-linking is started by adding BDDE. Dynamiccross-linking allows a product to be obtained that macroscopically has ahomogeneous appearance, and microscopically has a heterogenousappearance. This technique allows what is called a CohesivePolydensified Matrix to be obtained.

Placed in the presence of 1 ml of water for 2 minutes, the gel remainscohesive, which is not the case for biphasic products in which the“microparticle” component appears immediately. This product is availableunder the tradename Esthe'lis®.

The visco-elastic properties of Esthe'lis® make it a substance thatsculpts well in the tissue, with very gentle massage to position itcorrectly. It does not leave an “implanted cord” feeling and can even bereferred to as having a lifting effect.

In a preferred embodiment of the subject invention, hyaluronic acid isincorporated into lipid vesicles in order to administer hyaluronic acidto the skin of a patient. As described herein, any lipid vesiclesuitable for encapsulating hyaluronic acid and for administering to theskin of a human subject may be used.

Advantageously, when used according to the subject invention, thisproduct can have a hydrating effect in the treated area with, forexample, abolition of crow's feet during treatment of the eyes. Thenasolabial folds are on the areas treated on the cheeks.

Collagen

The compositions of the invention can include one or more purified, orrecombinant, collagens and/or collagen derivatives, or a combinationthereof. Collagen proteins useful in the invention include any nativecollagen proteins obtained from animal (e.g., human) cells and tissue,recombinantly expressed human collagen proteins (including fragments ofthe full-length collagen), and combinations and/or formulations thereof.

Purified collagens for use in the methods and compositions of theinvention may be isolated from animal or human tissues; however, the useof human collagen in the compositions and methods of the invention ispreferred when the subject to be treated is a human in order to preventan immune response to the collagen material. Collagen that is extractedfrom its source material (e.g., animal placenta, bone, hide, tendon) istypically a mixture of collagen type I with some collagen type III.Collagen material recovered from placenta, for example, is biased as tocollagen type and not entirely homogenous. Techniques for isolatingcollagen from human placentas are described in U.S. Pat. Nos. 5,002,071and 5,428,022.

In addition to employing collagen obtained directly from naturalsources, the methods and compositions of the invention include manydifferent types of collagen derivatives. Collagen derivatives may varyfrom naturally-occurring collagens in several respects. Collagenderivatives may be non-glycosylated or glycosylated differently thannaturally-occurring collagens. Desired glycosylation patterns may beproduced by a variety of methods, including direct chemical modificationand enzymatically catalyzed glycosylation and deglycosylation reactions.Desired glycosylation patterns may also be produced by inhibiting ordeleting enzymes necessary for producing the naturally-occurringglycosylation patterns found on collagens.

Collagen derivatives also include various fragments ofnaturally-occurring collagens. Such collagen fragments may be producedby, among other methods, chemically or enzymatically cleaving one ormore peptide bonds. Collagen derivatives may also contain one or moreamino acid residue differences as compared with corresponding amino acidresidue positions in a naturally-occurring collagen. Collagenderivatives containing such amino acid residue substitutions may beproduced by a variety of methods including genetic engineeringtechniques and by in vitro peptide synthesis. Additional collagenderivatives may be produced by varying the amount of hydroxylysinesand/or hydroxyprolines present in a given molecule, by the variedexpression of lysine hydroxylases, and/or proline hydroxylases, whereinthe hydroxylase genes (recombinant or otherwise) are also expressed in ahost cell for the expression of recombinant collagen, or derivativesthereof.

In certain preferred embodiments of the subject invention, purerecombinant collagen (e.g., types I or III) as opposed to the varioustypes found in animal (e.g., bovine) collagens can be used. Pure formsof recombinant collagen have performance characteristics that in someapplications are preferred to those from animal mixtures. A descriptionof how to produce collagen types I-III by recombinant DNA technology canbe found, among other places, in U.S. Pat. Nos. 5,405,757; 5,593,859;6,617,431; 6,428,978; PCT-published patent applications WO 93/07889 andWO94/16570, and related patents and applications. The recombinantproduction techniques described in these references may be readilyadapted so as to produce many different types of collagens, human orotherwise. Because an immune response can be elicited against non-humancollagen material, human collagens produced by recombinant DNAtechnology are preferably used in compositions and methods of thesubject invention.

A preferred collagen for use in the invention, for example, isrecombinant human collagen expressed in and purified from humanfibroblasts. This collagen material is produced by Inamed Corporation(Santa Barbara, Calif.) and is sold under the trade names CosmoDerm® andCosmoPlast®. Fibroblast cells used for culturing collagen in this methodare screened for known pathogens and the resultant collagen material istested for contaminants. Once the collagen is isolated from the cells,it is subjected to viral inactivation for increased safety. Methods forexpressing recombinant genes in human fibroblasts are well known in theart.

Two additional collagen materials that are produced using recombinantDNA technology and that can be used in the invention are FG-5017 andFG-5016 (FibroGen, South San Francisco, Calif.). FG-5017 is made ofrecombinant human collagen type III formulated for safety and efficacyas an injectible gel. FG-5016 is a recombinant human collagen type III(rhCIII) developed to replace animal-derived collagen in a variety ofpharmaceutical and medical device applications. FG-5016 is a highlypurified and fully characterized biomaterial, which is produced usingrecombinant methodology in a yeast expression system free of animalcomponents. This methodology involves the coordinate expression of genesencoding collagen and encoding prolyl 4-hydroxylase which enablesformation of thermally stable, triple helical collagen.

Methods for expressing recombinant genes in yeast cells are well knownin the art and are described in many references including Romanos etal., Yeast 8:423-488, 1992; Cohen et al., Nature 366: 698-701, 1993; andA. Wiseman. Genetically Engineered Proteins and Enzymes From Yeast:Production Control, 1991, Ellis Norwood Press, New York. Methods forproducing triple helical collagen in yeast cells are described in U.S.Pat. No. 6,451,557. Triple helical protein products produced in theyeast cells can be purified from the cells by techniques well known inthe art including standard chromatographic and precipitation techniques(See, e.g., Miller and Rhodes, Meth Enzymol., 82:33-64, 1982; and R. L.Trelstad, Native Collagen Fractionation, In: Immunochemistry of theExtracellular Matrix, vol. 1, H. Furthmayr, ed., CRC Press, Boca Raton,Fla., 1982, p. 31-41).

Regardless of the collagen source, preferred collagen and collagenderivatives for use in the invention are those that are sized to fitwithin the lipid vesicles of the invention, e.g., less than about 800nm. Because collagen fibrils are 20-150 nm in size, fibrils rather thanfibers (which are 1000-50,000 nm) are preferred. To maintain collagen inthe fibril form, the pH and/or ionic strength of the solution containingthe fibrils can be appropriately manipulated. A number of methods existto reduce collagen size, including an enzymatic breakdown using aprotease. Collagen can also be broken down mechanically. For example,collagen can be processed mechanically after drying to produce fineparticles that are less than 800 nm in size. Additionally, extensivehydrolysis of a collagen-containing solution may be used to preventfiber formation.

Lipid Vesicles Containing Collagen and/or Hyaluronic Acid

The invention provides compositions including lipid vesiclesincorporating human collagen (and/or a fragment thereof) or a collagenderivative, and/or hyaluronic acid. The vesicles containing the activeagent(s) are useful for administering the active agent(s) to a subject.Any lipid vesicle suitable for encapsulating collagen and/or hyaluronicacid, and for administering to the skin of a human subject may be used.

Vesicles of the invention are vesicles having one or more lipid bilayermembranes surrounding a cavity. Lipid vesicles for use in the inventionare typically in the range of about 50 to about 950 um (e.g., 50, 100,200, 300, 400, 500, 600, 700, 800, 900, 950 nm) in size. Methods forproducing and using lipid vesicles are well known in the art and aredescribed, e.g., in U.S. Pat. Nos. 4,917,951 and 5,013,497; Walde P. andIchikawa S., Biomol Eng., 18:143-177, 2001; Hunter D. G. and Frisken B.J., Biophys J., 74:2996-3002, 1998; and Cevc G., Adv Drug Deliv Rev.,56:675-711, 2004.

Collagen to be encapsulated within lipid vesicles can be any suitableform, e.g., a preparation of collagen type I, collagen type III, amixture of collagen type I and collagen type III, a collagen derivative,or a combination thereof.

Hyaluronic acid may be as described above. Vesicles may contain onlycollagen or only HA, or a combination thereof. The compositions of thesubject invention may comprise vesicles that contain only one activeagent, or multiple active agents.

The lipid vesicles of the invention can include non-phospholipidsurfactants. They can also include a charge-producing agent and atargeting molecule. Thus, vesicles made of non-phospholipid “membranemimetic” amphiphiles are useful in the invention. These are moleculesthat have a hydrophilic head group attached to a hydrophobic tail andinclude long-chain fatty acids, long-chain alcohols and derivatives,long-chain amino and glycerolipids. In the bilayers, the fatty acidtails point into the membrane's interior and the polar head groups pointoutward. The polar groups at one surface of the membrane point towardsthe vesicle's interior and those at the other surface point toward theexternal environment. As a vesicle forms during its manufacture, anywater-soluble molecules that have been added to the water areincorporated into the aqueous spaces in the interior of the spheres,whereas any lipid-soluble molecules added during vesicle formation areincorporated into the core of the vesicles.

Paucilamellar vesicles that can be formed from many bio-compatible,single-tailed amphiphiles are preferred for use in the invention. Suchpaucilamellar lipid vesicles include non-phospholipid vesicles havingone or several lipid bilayer membranes surrounding a large amorphouscore in which a chemical entity of interest (i.e., collagen and/or HA)is encapsulated.

Non-phospholipid paucilamellar lipid vesicles are sold under the tradename Novasome® (IGI Inc., Buena, N.J.). Several Novasome' formulationsexist (e.g., Novasome®A, Novasome® D), Novasome® Day Cream).

Novasome® vesicles are useful for encapsulating chemical ingredients toaid in formulation, increase delivery to site of action and stabilizechemical ingredients in the formulation. These lipid vesicles aregenerally about 200-700 nanometers in size, depending upon a widevariety of membrane constituents individually chosen for each particularpurpose. Their size distribution is uniform, and encapsulationefficiency can be nearly 100% for lipid cargo and 85% for aqueousmaterials. Finely divided insoluble particles (e.g., insolublepharmaceuticals) can also be encapsulated.

Novasome® vesicles are inherently stable, and can be tailored to bestable at pH levels ranging from 2-13 as well as temperature ranges aslow as liquid nitrogen to above the boiling point of water. They can bestable to solvents including alcohols, ethers, esters, gasoline, dieseland other fuels. They can encapsulate fragrances and flavors whichcontain volatile and fragile ethers, esters, aldehydes, etc. Thesevesicles can release their cargo under varying physical and chemicalcircumstances including heat, light, pH changes, enzymatic degradation,drying transmembrane diffusion, etc.

Protocols for producing and administering Novasome® formulations aredescribed, for example, in U.S. Pat. Nos. 4,855,090; 4,911,928;5,474,848; 5,628,936; 6,387,373; Holick et al., British Journal ofDermatology 149:1365-2133, 2003; Gupta et al., Vaccine 14:219-225, 1996;and Wallach D F H and Philippot J., New Type of Lipid Vesicle: Novasome™In: Liposome Technology, 2″ ed., Gregorriadis G., CRC Press, Boca Raton,Fla., 1982, pp. 141-151; Niemiec et al., Pharmaceutical Research12:1184-1188, 1995; and Alfieri D R, Cosmetic Dermatology 10:42-52,1997.

In one embodiment, the liposomes are those used in “Day Cream.”

In certain embodiments of the subject invention, the lipid vesicles(e.g., non-phospholipid paucilamellar lipid vesicles) may also includetargeting molecules, either hydrophilic or amphiphilic, which can beused to direct the vesicles to a particular target in order to allowrelease of the HA, collagen or collagen derivative from within thevesicle at a specified biological location. If hydrophilic targetingmolecules are used, they can be coupled directly or via a spacer to anOH residue of the polyoxyethylene portion of the surfactant, or they canbe coupled, using techniques in the art, to molecules such as palmiticacid, long chain amines, or phosphatidyl ethanolamine. If spacers areused, the targeting molecules can be interdigitated into the hydrophiliccore of the bilayer membrane via the acyl chains of these compounds.Preferred hydrophilic targeting molecules include monoclonal antibodies,other immunoglobulins, lectins, and peptide hormones.

In addition to hydrophilic targeting molecules, it is also possible touse amphiphilic targeting molecules. Amphiphilic targeting molecules arenormally not chemically coupled to the surfactant molecules but ratherinteract with the lipophilic or hydrophobic portions of the moleculesconstituting the bilayer lamellae of the lipid vesicles. Preferredamphiphilic targeting molecules are neutral glycolipids,galactocerebrosides (e.g., for hepatic galactosyl receptors), or chargedglycolipids such as gangliosides.

In some embodiments, charge-producing materials and steroids such ascholesterol or hydrocortisone or their analogues and derivatives areused in the formation of the lipid vesicles (e.g., paucilamellar lipidvesicles). Preferred charge-producing materials include negativecharge-producing materials such as dicetyl phosphate, cetyl sulphate,phosphatidic acid, phosphatidyl serine, oleic acid, palmitic acid, ormixtures thereof. In order to provide a net positive charge to thevesicles, long chain amines, e.g., stearyl amines or oleyl amines, longchain pyridinium compounds, e.g., cetyl pyridinium chloride, quaternaryammonium compounds, or mixtures of these can be used. Another example ofa positive charge-producing material is hexadecyl trimethylammoniumbromide, a potent disinfectant.

Preparing Lipid Vesicles

Lipid vesicles used according to the subject invention can be any of alarge variety of lipid vesicles known in the art and can be madeaccording to any of a large number of production methods. Materials andprocedures for forming lipid vesicles are well-known to those skilled inthe art. In general, lipids or lipophilic substances are dissolved in anorganic solvent. When the solvent is removed, such as under vacuum byrotary evaporation, the lipid residue forms a film on the wall of thecontainer. An aqueous solution that typically contains electrolytes orhydrophilic biologically agent materials is then added to the film.Large multilamellar vesicles are produced upon agitation. When smallermultilamellar vesicles are desired, the larger vesicles are subjected tosonication, sequential filtration through filters with decreasing poresize or reduced by other forms of mechanical shearing. Lipid vesiclescan also take the form of unilamellar vesicles, which are prepared bymore extensive sonication of multilamellar vesicles, and consist of asingle spherical lipid bilayer surrounding an aqueous solution. Acomprehensive review of all the aforementioned lipid vesicles andmethods for their preparation are described in “Liposome Technology”,ed. G. Gregoriadis, CRC Press Inc., Boca Raton, Fla., Vol. I, II & III(1984). For methods of preparing lipid vesicles, also see U.S. Pat. Nos.4,485,054, 4,761,288, 5,013,497, 5,653,996, and 6,855,296.

To prepare non-phospholipid paucilamellar lipid vesicles formed ofnon-phospholipid surfactant material and containing an aqueous-basedcollagen and/or HA material, any suitable method known in the art can beused. Methods of preparing non-phospholipid paucilamellar lipid vesiclestypically involve first forming a lipophilic phase by combining severallipophilic components including surfactant material and then heating andblending this mixture. Examples of suitable surfactant materials includebut are not limited to polyoxyethylene (2) cetyl ether, polyoxyethylene(4) lauryl ether, glyceryl monostearate, and poly oxyethylene (9)glyceryl stearate. The resultant lipophilic phase is then blended withan aqueous phase having an aqueous buffer and an aqueous solublecollagen formulation, under shear mixing conditions, to form thepaucilamellar lipid vesicles. In this method, the temperature of thelipophilic phase is elevated in order to make it flowable followed bycarrying out the shear mixing between the lipophilic phase and theaqueous phase at a temperature such that both phases are liquids. Whileit is often desirable to use the same temperature for both phases, thisis not always necessary. Any other method known to the skilled artisancan also be used. Preferred methods for making the paucilamellar lipidvesicles of the invention are described in U.S. Pat. No. 4,911,928.

To encapsulate oil-based collagen or collagen-containing formulationswithin paucilamellar lipid vesicles, the collagen or collagen-containingformulation is dispersed in an oil or wax forming an oily phase. The oilor wax is a water immiscible oily solution selected from a groupconsisting of oils, waxes, natural and synthetic triglycerides, acylesters, and petroleum derivatives, and their analogues and derivatives.The oily phase containing the oil-dispersible material is mixed with thelipid phase and the combined oil-lipid phase is blended under shearmixing conditions with the aqueous phase. Surfactants useful in theencapsulation process are the same as those used to make paucilamellarlipid vesicles with an aqueous core.

Paucilamellar lipid vesicles can be made by a variety of devices whichprovide sufficiently high shear for shear mixing. Many such devices areavailable on the market including a Microfluidizer® such as is made byMicroFluidics Corp. (Newton, Mass.), a “French”-type press, or someother device which provides a high enough shear force and the ability tohandle heated, semiviscous lipids. If a very high shear device is used,it may be possible to microemulsify powdered lipids, under pressure, ata temperature below their normal melting points and still form thecollagen-containing paucilamellar lipid vesicles of the presentinvention.

A device which is particularly useful for making the paucilamellar lipidvesicles of the present invention has been developed by Micro VesicularSystems, Inc., (Vineland, N.J.) and is further described in U.S. Pat.No. 4,895,452. Briefly, this device has a substantially cylindricalmixing chamber with at least one tangentially located inlet orifice. Oneor more orifices lead to a reservoir for the lipophilic phase, mixedwith an oil phase if lipid-core paucilamellar lipid vesicles are to beformed, and at least one of the other orifices is attached to areservoir for the aqueous phase. The different phases are driven intothe cylindrical chamber through pumps, e.g., positive displacementpumps, and intersect in such a manner as to form a turbulent flow withinthe chamber. The paucilamellar lipid vesicles form rapidly, e.g., lessthan 1 second, and are removed from the chamber through an axiallylocated discharge orifice. Preferably, there are four tangentiallylocated inlet orifices and the lipid and aqueous phases are drawn fromreservoirs, through positive displacement pumps, to alternatingorifices. The fluid stream through the tangential orifices is guided ina spiral flow path from each inlet or injection orifice to the dischargeorifice. The flow paths are controlled by the orientation or placementof the inlet or injection orifices so as to create a mixing zone by theintersection of the streams of liquid. The pump speeds, as well as theorifice and feed line diameters, are selected to achieve proper shearmixing for lipid vesicle formation. In most circumstances, turbulentflow is selected to provide adequate mixing.

No matter what device is used to form the paucilamellar lipid vesicles,if proper shear mixing is achieved they have a structure involving alarge, unstructured amorphous center surrounded by a plurality of lipidbilayers having aqueous layers interspersed there between. About fourlipid bilayers is standard with 2-8 possible. The amorphous center maybe entirely filled with an aqueous material, e.g., a buffer and anyaqueous material to be encapsulated, or may be partially or totallyfilled with an oily material, forming lipid-core paucilamellar lipidvesicles. If an aqueous center is used, the paucilamellar lipid vesicleswill normally range in diameter from about 0.5-2μ while if an oilycenter is used, the size may increase to up to about 15-20μ dependingupon the amount of oil used.

Use of Cyclodextin as a Carrier

Additionally, cyclodextrins are an alternate option for a collagenand/or HA carrier system into the dermis of the skin. Cyclodextrins arecomplex carbohydrates of 6, 7, or 8 D-glucopyranose residues that arelinked by 1,4 glycosidic bonds. The three forms are dependent on thenumber of D-glucopyranose residues, the alpha form having 6, beta having7, and gamma having 8. The alpha structure forms an annular ring with aninternal hydrophobic cavity and a hydrophilic outer surface. Eachcyclodextrin associates with a guest compound by fitting the compoundinto the hydrophobic cavity forming an inclusion complex. In this waycyclodextrins can be used as a delivery system to deliver a desiredamount of material to a target location.

In one embodiment hydroxypropyl beta cyclodextrins can be used.Cyclodextrins are used because they have the ability to alter thephysical, chemical, and biological properties of an associated guestcompound through formation of the inclusion complex. This complexenhances the solubility, stability, and bioavailability of the guestcompound so that the material can be isolated and used in a controlleddelivery system. Formation an inclusion complex of collagen with analpha-cyclodextrin allows for a targeted delivery system to the dermis.

The principal method for the isolation and purification ofalpha-cyclodextrin takes advantage of its complex-forming ability. Atcompletion of the reaction, 1-decanol is added to the reaction mixtureto form an insoluble 1:1 alpha-cyclodextrin: 1-decanol inclusioncomplex. The complex is continuously mixed with water and separated fromthe reaction mixture by centrifugation. The recovered complex isre-suspended in water and dissolved by heating. Subsequent cooling leadsto re-precipitation of the complex. The precipitate is recovered bycentrifugation, and 1-decanol is removed by steam distillation. Uponcooling, alpha-cyclodextrin crystallizes from solution. The crystals areremoved by filtration and dried, yielding a white crystalline powderwith a water content under 11%. The purity on a dried basis is at least98%.

Dermatologically-Acceptable Carrier

The topical compositions of the present invention, in addition to thevesicle-contained collagen and/or HA, can further comprise adermatologically acceptable carrier. A safe and effective amount ofcarrier is typically from about 50% to about 99.99%, preferably fromabout 80% to about 99.9%, more preferably from about 90% to about 98%,and even more preferably from about 90% to about 95% of the composition.

The carrier can be in a wide variety of forms. For example, emulsioncarriers, including, but not limited to, oil-in-water, water-in-oil,water-in-oil-in-water, and oil-in-water-in-silicone emulsions, areuseful herein.

Emulsions according to the present invention can contain a solution asdescribed above and a lipid or oil. Lipids and oils may be derived fromanimals, plants, or petroleum and may be natural or synthetic (i.e.,man-made). Preferred emulsions also contain a humectant, such asglycerin. Emulsions will preferably further contain from about 0.01% toabout 10%, more preferably from about 0.1% to about 5%, of anemulsifier, based on the weight of the carrier. Emulsifiers may benonionic, anionic or cationic. Suitable emulsifiers are disclosed in,for example, U.S. Pat. No. 3,755,560, issued Aug. 28, 1973 to Dickert etal.; U.S. Pat. No. 4,421,769, issued Dec. 20, 1983 to Dixon et al.; andMcCutcheon's Detergents and Emulsifiers, North American Ed., pages317-324 (1986).

The emulsion may also contain an anti-foaming agent to minimize foamingupon application to the epidermal tissue. Anti-foaming agents includehigh molecular weight silicones and other materials well known in theart for such use.

Suitable emulsions may have a wide range of viscosities, depending onthe desired product form. Exemplary low viscosity emulsions, which arepreferred, have a viscosity of about 50 centistokes or less, morepreferably about 10 centistokes or less, still more preferably about 5centistokes or less.

Water-in-silicone emulsions can contain a continuous silicone phase anda dispersed aqueous phase. The continuous silicone phase exists as anexternal phase that contains or surrounds the discontinuous aqueousphase described hereinafter. The continuous silicone phase may containone or more non-silicone oils. Examples of non-silicone oils suitablefor use in the continuous silicone phase are those well known in thechemical arts in topical personal care products in the form ofwater-in-oil emulsions, e.g., mineral oil, vegetable oils, syntheticoils, semisynthetic oils, etc.

In emulsion technology, the term “dispersed phase” is a term well-knownto one skilled in the art that means that the phase exists as smallparticles or droplets that are suspended in and surrounded by acontinuous phase. The dispersed phase is also known as the internal ordiscontinuous phase. The dispersed aqueous phase is a dispersion ofsmall aqueous particles or droplets suspended in and surrounded by thecontinuous silicone phase described hereinbefore.

The aqueous phase can be water, or a combination of water and one ormore water soluble or dispersible ingredients. Nonlimiting examples ofsuch ingredients include thickeners, acids, bases, salts, chelants,gums, water-soluble or dispersible alcohols and polyols, buffers,preservatives, sunscreening agents, colorings, and the like.

Water-in-silicone emulsions can contain an emulsifier. In oneembodiment, the composition contains from about 0.1% to about 10%emulsifier, more preferably from about 0.5% to about 7.5%, still morepreferably from about 1% to about 5%, emulsifier by weight of thecomposition. The emulsifier helps disperse and suspend the aqueous phasewithin the continuous silicone phase.

Other topical carriers include oil-in-water emulsions, having acontinuous aqueous phase and a hydrophobic, water-insoluble phase (“oilphase”) dispersed therein. Examples of suitable oil-in-water emulsioncarriers are described in U.S. Pat. No. 5,073,371, to Turner, D. J. etal., issued Dec. 17, 1991, and U.S. Pat. No. 5,073,372, to Turner, D. J.et al., issued Dec. 17, 1991.

An oil-in-water emulsion can contain a structuring agent to assist inthe formation of a liquid crystalline gel network structure. Structuringagents include stearic acid, palmitic acid, stearyl alcohol, cetylalcohol, behenyl alcohol, stearic acid, palmitic acid, the polyethyleneglycol ether of stearyl alcohol having an average of about 1 to about 21ethylene oxide units, the polyethylene glycol ether of cetyl alcoholhaving an average of about 1 to about 5 ethylene oxide units, andmixtures thereof.

In certain embodiments, oil-in-water emulsions that contain at least onehydrophilic surfactant which can disperse the hydrophobic materials inthe water phase (percentages by weight of the topical carrier). Thesurfactant, at a minimum, must be hydrophilic enough to disperse inwater. Among the nonionic surfactants that are useful herein are thosethat can be broadly defined as condensation products of long chainalcohols, e.g. C₈₋₃₀ alcohols, with sugar or starch polymers, i.e.,glycosides.

Other suitable surfactants useful herein include a wide variety ofcationic, anionic, zwitterionic, and amphoteric surfactants such as areknown in the art. See, e.g., McCutcheon's, Detergents and Emulsifiers,North American Edition (1986), published by Allured PublishingCorporation; U.S. Pat. No. 5,011,681 to Ciotti et al., issued Apr. 30,1991; U.S. Pat. No. 4,421,769 to Dixon et al., issued Dec. 20, 1983; andU.S. Pat. No. 3,755,560 to Dickert et al., issued Aug. 28, 1973; thesefour references are incorporated herein by reference in their entirety.The hydrophilic surfactants useful herein can contain a singlesurfactant, or any combination of suitable surfactants. The exactsurfactant (or surfactants) chosen will depend upon the pH of thecomposition and the other components present.

Also useful herein are cationic surfactants, such as dialkyl quaternaryammonium compounds, examples of which are described in U.S. Pat. Nos.5,151,209; 5,151,210; 5,120,532; 4,387,090; 3,155,591; 3,929,678;3,959,461; McCutcheon's, Detergents & Emulsifiers, (North Americanedition 1979) M.C. Publishing Co.; and Schwartz, et al., Surface ActiveAgents, Their Chemistry and Technology, New York: IntersciencePublishers, 1949; which descriptions are incorporated herein byreference.

A wide variety of anionic surfactants are also useful herein. See, e.g.,U.S. Pat. No. 3,929,678, to Laughlin et al., issued Dec. 30, 1975, whichis incorporated herein by reference in its entirety. Nonlimitingexamples of anionic surfactants include the alkoyl isethionates, and thealkyl and alkyl ether sulfates.

Examples of amphoteric and zwitterionic surfactants are those which arebroadly described as derivatives of aliphatic secondary and tertiaryamines in which the aliphatic radical can be straight or branched chainand wherein one of the aliphatic substituents contains from about 8 toabout 22 carbon atoms (preferably C₈-C₁₈) and one contains an anionicwater solubilizing group, e.g., carboxy, sulfonate, sulfate, phosphate,or phosphonate.

The topical compositions of the subject invention, including but notlimited to lotions and creams, may contain a dermatologically acceptableemollient. Such compositions preferably contain from about 1% to about50% of the emollient. As used herein, “emollient” refers to a materialuseful for the prevention or relief of dryness, as well as for theprotection of the skin. A wide variety of suitable emollients are knownand may be used herein. Sagarin, Cosmetics, Science and Technology, 2ndEdition, Vol. 1, pp. 32-43 (1972), incorporated herein by reference,contains numerous examples of materials suitable as an emollient. Apreferred emollient is glycerin. Glycerin is preferably used in anamount of from or about 0.001 to or about 30%, more preferably from orabout 0.01 to or about 20%, still more preferably from or about 0.1 toor about 10%, e.g., 5%.

Creams are generally thicker than lotions due to higher levels ofemollients or higher levels of thickeners.

Ointments of the present invention may contain a simple carrier base ofanimal or vegetable oils or semi-solid hydrocarbons (oleaginous);absorption ointment bases which absorb water to form emulsions; or watersoluble carriers, e.g., a water soluble solution carrier. Ointments mayfurther contain a thickening agent, such as described in Sagarin,Cosmetics, Science and Technology, 2nd Edition, Vol. 1, pp. 72-73(1972), incorporated herein by reference, and/or an emollient. Forexample, an ointment may contain from about 2% to about 10% of anemollient; from about 0.1% to about 2% of a thickening agent; and thevesicle-collagen in the above described amounts.

Additional Skin Care Agents

The compositions of the present invention may contain one or moreadditional skin care agents, in addition to collagen and/or HA, theagents enumerated below do not include water unless specifically stated.

The additional agents should be suitable for application to epidermaltissue, that is, when incorporated into the composition they aresuitable for use in contact with human epidermal tissue without unduetoxicity, incompatibility, instability, allergic response, and the like.The CTFA Cosmetic Ingredient Handbook, Second Edition (1992) describes awide variety of nonlimiting cosmetic and pharmaceutical ingredientscommonly used in the skin care industry, which are suitable for use inthe compositions of the present invention.

Examples of such ingredient classes include: abrasives, absorbents,aesthetic components such as fragrances, pigments, colorings/colorants,essential oils, skin sensates, astringents, etc. (e.g., clove oil,menthol, camphor, eucalyptus oil, eugenol, menthyl lactate, witch hazeldistillate), anti-acne agents, anti-caking agents, antifoaming agents,antimicrobial agents (e.g., iodopropyl butylcarbamate), antioxidants,binders, biological additives, buffering agents, bulking agents,chelating agents, chemical additives, colorants, cosmetic astringents,cosmetic biocides, denaturants, drug astringents, external analgesics,film formers or materials, e.g., polymers, for aiding the film-formingproperties and substantivity of the composition (e.g., copolymer ofeicosene and vinyl pyrrolidone), opacifying agents, pH adjusters,propellants, reducing agents, sequestrants, skin bleaching andlightening agents (e.g., hydroquinone, kojic acid, ascorbic acid,magnesium ascorbyl phosphate, ascorbyl glucosamine), skin-conditioningagents (e.g., humectants, including miscellaneous and occlusive), skinsoothing and/or healing agents (e.g., panthenol and derivatives (e.g.,ethyl panthenol), aloe vera, pantothenic acid and its derivatives,allantoin, bisabolol, and dipotassium glycyrrhizinate), skin treatingagents, thickeners, and vitamins and derivatives thereof.

In any embodiment of the present invention, however, the agents usefulherein can be categorized by the benefit they provide or by theirpostulated mode of action. However, it is to be understood that theadditional agents for use herein can in some instances provide more thanone benefit or operate via more than one mode of action. Therefore,classifications herein are made for the sake of convenience and are notintended to limit the agent to that particular application orapplications listed.

Desquamation Agents

A safe and effective amount of a desquamation agent may be added to thecompositions of the present invention, more preferably from about 0.1%to about 10%, even more preferably from about 0.2% to about 5%, alsopreferably from about 0.5% to about 4%, by weight of the composition.Desquamation agents enhance the skin appearance benefits of the presentinvention. For example, the desquamation agents tend to improve thetexture of the skin (e.g., smoothness). One desquamation system that issuitable for use herein contains sulfhydryl compounds and zwitterionicsurfactants and is described in U.S. Pat. No. 5,681,852, to Bissett,incorporated herein by reference. Another desquamation system that issuitable for use herein contains salicylic acid and zwitterionicsurfactants and is described in U.S. Pat. No. 5,652,228 to Bissett,incorporated herein by reference. Zwitterionic surfactants such asdescribed in these applications are also useful as desquamatory agentsherein, with cetyl betaine being particularly preferred.

Anti-Acne Agents

The compositions of the present invention may contain a safe andeffective amount of one or more anti-acne agents. Examples of usefulanti-acne agents include resorcinol, sulfur, salicylic acid, benzoylperoxide, erythromycin, zinc, etc. Further examples of suitableanti-acne agents are described in further detail in U.S. Pat. No.5,607,980, issued to McAtee et al, on Mar. 4, 1997.

Anti-Wrinkle Agents/Anti-Atrophy Agents

The compositions of the present invention may further contain a safe andeffective amount of one or more anti-wrinkle agents or anti-atrophyagents. Exemplary anti-wrinkle/anti-atrophy agents suitable for use inthe compositions of the present invention include sulfur-containing Dand L amino acids and their derivatives and salts, particularly theN-acetyl derivatives, a preferred example of which isN-acetyl-L-cysteine; thiols, e.g. ethane thiol; hydroxy acids (e.g.,alpha-hydroxy acids such as lactic acid and glycolic acid orbeta-hydroxy acids such as salicylic acid and salicylic acid derivativessuch as the octanoyl derivative), phytic acid, lipoic acid;lysophosphatidic acid, skin peel agents (e.g., phenol and the like),vitamin B₃ compounds, retinoids, and hyaluronic acid, which enhance theepidermal tissue appearance benefits of the present invention,especially in regulating epidermal tissue condition, e.g., skincondition.

Vitamin B₃ Compounds

The compositions of the present invention may contain a safe andeffective amount of a vitamin B₃ compound. Vitamin B₃ compounds areparticularly useful for regulating skin condition as described in U.S.application Ser. No. 08/834,010, filed Apr. 11, 1997 (corresponding tointernational publication WO 97/39733 A1, published Oct. 30, 1997).Examples of suitable vitamin B₃ compounds are well known in the art andare commercially available from a number of sources, e.g., the SigmaChemical Company (St. Louis, Mo.); ICN Biomedicals, Inc. (Irvin, Calif.)and Aldrich Chemical Company (Milwaukee, Wis.). The vitamin compoundsmay be included as the substantially pure material, or as an extractobtained by suitable physical and/or chemical isolation from natural(e.g., plant) sources.

Retinoids

The compositions of the present invention may also contain a retinoid.As used herein. “retinoid” includes all natural and/or synthetic analogsof Vitamin A or retinol-like compounds which possess the biologicalactivity of Vitamin A in the skin as well as the geometric isomers andstereoisomers of these compounds. The retinoid is preferably retinol,retinol esters (e.g., C₂-C₂₂ alkyl esters of retinol, including retinylpalmitate, retinyl acetate, retinyl propionate), retinal, and/orretinoic acid (including all-trans retinoic acid and/or 13-cis-retinoicacid), more preferably retinoids other than retinoic acid. Thesecompounds are well known in the art and are commercially available froma number of sources, e.g., Sigma Chemical Company (St. Louis, Mo.), andBoerhinger Mannheim (Indianapolis, Ind.). Other retinoids which areuseful herein are described in U.S. Pat. No. 4,677,120, issued Jun. 30,1987 to Parish et al.; U.S. Pat. No. 4,885,311, issued Dec. 5, 1989 toParish et al.; U.S. Pat. No. 5,049,584, issued Sep. 17, 1991 to Purcellet al.; U.S. Pat. No. 5,124,356, issued Jun. 23, 1992 to Purcell et al.;and U.S. Pat. No. Reissue 34,075, issued Sep. 22, 1992 to Purcell et al.Other suitable retinoids are tocopheryl-retinoate [tocopherol ester ofretinoic acid (trans- or cis-), adapalene{6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid}, and tazarotene(ethyl 612-(4,4-dimethylthiochroman-6-yl)-ethynyl]nicotinate). Preferredretinoids are retinol, retinyl palmitate, retinyl acetate, retinylpropionate, retinal and combinations thereof.

Hydroxy Acids

The compositions of the present invention may contain a safe andeffective amount of a hydroxy acid. Preferred hydroxy acids for use inthe compositions of the present invention include salicylic acid andsalicylic acid derivatives.

Anti-Oxidants/Radical Scavengers

The compositions of the present invention may include a safe andeffective amount of an anti-oxidant/radical scavenger. Theanti-oxidant/radical scavenger is especially useful for providingprotection against UV radiation which can cause increased scaling ortexture changes in the stratum corneum and against other environmentalagents which can cause skin damage.

A safe and effective amount of an anti-oxidant/radical scavenger may beadded to the compositions of the subject invention, preferably fromabout 0.1% to about 10%, more preferably from about 1% to about 5%, ofthe composition.

Anti-oxidants/radical scavengers such as ascorbic acid (vitamin C) andits salts, ascorbyl esters of fatty acids, ascorbic acid derivatives(e.g., magnesium ascorbyl phosphate, sodium ascorbyl phosphate, ascorbylsorbate), tocopherol (vitamin E), tocopherol sorbate, tocopherolacetate, other esters of tocopherol, butylated hydroxy benzoic acids andtheir salts, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid(commercially available under the tradename Trolox®), gallic acid andits alkyl esters, especially propyl gallate, uric acid and its salts andalkyl esters, sorbic acid and its salts, lipoic acid, amines (e.g.,N,N-diethylhydroxylamine, amino-guanidine), sulfhydryl compounds (e.g.,glutathione), dihydroxy fumaric acid and its salts, lycine pidolate,arginine pilolate, nordihydroguaiaretic acid, bioflavonoids, curcumin,lysine, methionine, proline, superoxide dismutase, silymarin, teaextracts, grape skin/seed extracts, melanin, and rosemary extracts maybe used. Preferred anti-oxidants/radical scavengers are selected fromtocopherol sorbate and other esters of tocopherol, more preferablytocopherol sorbate. For example, the use of tocopherol sorbate intopical compositions and applicable to the present invention isdescribed in U.S. Pat. No. 4,847,071, issued on Jul. 11, 1989 to DonaldL. Bissett, Rodney D. Bush and Ranjit Chatterjee.

Chelators

The compositions of the present invention may also contain a safe andeffective amount of a chelator or chelating agent. As used herein,“chelator” or “chelating agent” means an active agent capable ofremoving a metal ion from a system by forming a complex so that themetal ion cannot readily participate in or catalyze chemical reactions.The inclusion of a chelating agent is especially useful for providingprotection against UV radiation which can contribute to excessivescaling or skin texture changes and against other environmental agentswhich can cause skin damage.

A safe and effective amount of a chelating agent may be added to thecompositions of the subject invention, preferably from about 0.1% toabout 10%, more preferably from about 1% to about 5%, of thecomposition. Exemplary chelators that are useful herein are disclosed inU.S. Pat. No. 5,487,884, issued Jan. 30, 1996 to Bissett et al.;International Publication No. 91/16035, Bush et al., published Oct. 31,1995; and International Publication No. 91/16034, Bush et al., publishedOct. 31, 1995. Preferred chelators useful in compositions of the subjectinvention are furildioxime, furilmonoxime, and derivatives thereof.

Flavonoids

The compositions of the present invention may optionally contain aflavonoid compound. Flavonoids are broadly disclosed in U.S. Pat. Nos.5,686,082 and 5,686,367, both of which are herein incorporated byreference. Flavonoids suitable for use in the present invention areflavanones selected from unsubstituted flavanones, mono-substitutedflavanones, and mixtures thereof; chalcones selected from unsubstitutedchalcones, mono-substituted chalcones, di-substituted chalcones,tri-substituted chalcones, and mixtures thereof; flavones selected fromunsubstituted flavones, mono-substituted flavones, di-substitutedflavones, and mixtures thereof; one or more isoflavones; coumarinsselected from unsubstituted coumarins, mono-substituted coumarins,di-substituted coumarins, and mixtures thereof; chromones selected fromunsubstituted chromones, mono-substituted chromones, di-substitutedchromones, and mixtures thereof; one or more dicoumarols; one or morechromanones; one or more chromanols; isomers (e.g., cis/trans isomers)thereof; and mixtures thereof. By the term “substituted” as used hereinmeans flavonoids wherein one or more hydrogen atom of the flavonoid hasbeen independently replaced with hydroxyl, C1-C8 alkyl, C1-C4 alkoxyl,0-glycoside, and the like or a mixture of these substituents.

Examples of suitable flavonoids include, but are not limited to,unsubstituted flavanone, mono-hydroxy flavanones (e.g., 2′-hydroxyflavanone, 6-hydroxy flavanone, 7-hydroxy flavanone, etc.), mono-alkoxyflavanones (e.g., 5-methoxy flavanone, 6-methoxy flavanone, 7-methoxyflavanone, 4′-methoxy flavanone, etc.), unsubstituted chalcone(especially unsubstituted trans-chalcone), mono-hydroxy chalcones (e.g.,2′-hydroxy chalcone, 4′-hydroxy chalcone, etc.), di-hydroxy chalcones(e.g., 2′,4-dihydroxy chalcone, 2′,4′-dihydroxy chalcone, 2,2′-dihydroxychalcone, 2′,3-dihydroxy chalcone, 2′,5′-dihydroxy chalcone, etc.), andtri-hydroxy chalcones (e.g., 2′,3′,4′-trihydroxy chalcone,4,2′,4′-trihydroxy chalcone, 2,2′,4′-trihydroxy chalcone, etc.),unsubstituted flavone, 7,2′-dihydroxy flavone, 3′,4′-dihydroxynaphthoflavone, 4′-hydroxy flavone, 5,6-benzoflavone, and7,8-benzoflavone, unsubstituted isoflavone, daidzein (7,4′-dihydroxyisoflavone), 5,7-dihydroxy-4′-methoxy isoflavone, soy isoflavones (amixture extracted from soy), unsubstituted coumarin, 4-hydroxy coumarin,7-hydroxy coumarin, 6-hydroxy-4-methyl coumarin, unsubstituted chromone,3-formyl chromone, 3-formyl-6-isopropyl chromone, unsubstituteddicoumarol, unsubstituted chromanone, unsubstituted chromanol, andmixtures thereof.

Preferred for use herein are unsubstituted flavanone, methoxyflavanones, unsubstituted chalcone, 2′,4-dihydroxy chalcone, andmixtures thereof. More preferred are unsubstituted flavanone,unsubstituted chalcone (especially the trans isomer), and mixturesthereof.

They can be synthetic materials or obtained as extracts from naturalsources (e.g., plants). The naturally sourced material can also furtherbe derivatized (e.g., an ester or ether derivative prepared followingextraction from a natural source). Flavonoid compounds useful herein arecommercially available from a number of sources, e.g., Indofine ChemicalCompany, Inc. (Somerville, N.J.), Steraloids, Inc. (Wilton, N.H.), andAldrich Chemical Company, Inc. (Milwaukee, Wis.).

Anti-Inflammatory Agents

A safe and effective amount of an anti-inflammatory agent may be addedto the compositions of the present invention, preferably from about 0.1%to about 10%, more preferably from about 0.5% to about 5%, of thecomposition. The anti-inflammatory agent enhances the skin appearancebenefits of the present invention, e.g., such agents contribute to amore uniform and acceptable skin tone or color. The exact amount ofanti-inflammatory agent to be used in the compositions will depend onthe particular anti-inflammatory agent utilized since such agents varywidely in potency.

Steroidal anti-inflammatory agents, including but not limited to,corticosteroids such as hydrocortisone, hydroxyltriamcinolone,alpha-methyl dexamethasone, dexamethasone-phosphate, beclomethasonedipropionates, clohetasol valerate, desonide, desoxymethasone,desoxycorticosterone acetate, dexamethasone, dichlorisone, diflorasonediacetate, diflucortolone valerate, fluadrenolone, flucloroloneacetonide, fludrocortisone, flumethasone pivalate, fluosinoloneacetonide, fluocinonide, flucortine butylesters, fluocortolone,fluprednidene (fluprednylidene) acetate, flurandrenolone, halcinonide,hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone,triamcinolone acetonide, cortisone, cortodoxone, flucetonide,fludrocortisone, difluorosone diacetate, fluradrenolone,fludrocortisone, difluorosone diacetate, fluradrenolone acetonide,medrysone, amcinafel, amcinafide, betamethasone and the balance of itsesters, chloroprednisone, chlorprednisone acetate, clocortelone,clescinolone, dichlorisone, diflurprednate, flucloronide, flunisolide,fluoromethalone, fluperolone, fluprednisolone, hydrocortisone valerate,hydrocortisone cyclopentylpropionate, hydrocortamate, mepredni sone,paramethasone, prednisolone, prednisone, beclomethasone dipropionate,triamcinolone, and mixtures thereof may be used. The preferred steroidalanti-inflammatory for use is hydrocortisone.

A second class of anti-inflammatory agents which is useful in thecompositions includes the nonsteroidal anti-inflammatory agents. Thevariety of compounds encompassed by this group are well-known to thoseskilled in the art. For detailed disclosure of the chemical structure,synthesis, side effects, etc. of non-steroidal anti-inflammatory agents,one may refer to standard texts, including Anti-inflammatory andAnti-Rheumatic Drugs, K. D. Rainsford, Vol. I-III, CRC Press, BocaRaton, (1985), and Anti-inflammatory Agents, Chemistry and Pharmacology,1, R. A. Scherrer, et al., Academic Press, New York (1974).

Specific non-steroidal anti-inflammatory agents useful in thecomposition invention include, but are not limited to:

1) the oxicams, such as piroxicam, isoxicam, tenoxicam, sudoxicam, andCP-14,304;

2) the salicylates, such as aspirin, disalcid, benorylate, trilisate,safapryn, solprin, diflunisal, and fendosal;

3) the acetic acid derivatives, such as diclofenac, fenclofenac,indomethacin, sulindac, tolmetin, isoxepac, furofenac, tiopinac,zidometacin, acemetacin, fentiazac, zomepirac, clindanac, oxepinac,felbinac, and ketorolac;

4) the fenamates, such as mefenamic, meclofenamic, flufenamic, niflumic,and tolfenamic acids;

5) the propionic acid derivatives, such as ibuprofen, naproxen,benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen,indopropfen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen,tioxaprofen, suprofen, alminoprofen, and tiaprofenic; and

6) the pyrazoles, such as phenylbutazone, oxyphenbutazone, feprazone,azapropazone, and trimethazone.

Mixtures of these non-steroidal anti-inflammatory agents may also beemployed, as well as the dermatologically acceptable salts and esters ofthese agents. For example, etofenamate, a flufenamic acid derivative, isparticularly useful for topical application. Of the nonsteroidalanti-inflammatory agents, ibuprofen, naproxen, flufenamic acid,etofenamate, aspirin, mefenamic acid, meclofenamic acid, piroxicam andfelbinac are preferred; ibuprofen, naproxen, ketoprofen, etofenamate,aspirin and flufenamic acid are more preferred.

Finally, so-called “natural” anti-inflammatory agents are useful inmethods of the present invention. Such agents may suitably be obtainedas an extract by suitable physical and/or chemical isolation fromnatural sources (e.g., plants, fungi, by-products of microorganisms) orcan be synthetically prepared. For example, candelilla wax, bisabolol(e.g., alpha bisabolol), aloe vera, plant sterols (e.g., phytosterol),Manjistha (extracted from plants in the genus Rubia, particularly RubiaCordifolia), and Guggal (extracted from plants in the genus Commiphora,particularly Commiphora Mukul), kola extract, chamomile, red cloverextract, and sea whip extract, may be used.

Additional anti-inflammatory agents useful herein include compounds ofthe Licorice (the plant genus/species Glycyrrhiza glabra) family,including glycyrrhetic acid, glycyrrhizic acid, and derivatives thereof(e.g., salts and esters). Suitable salts of the foregoing compoundsinclude metal and ammonium salts. Suitable esters includeC.sub.2-C.sub.24 saturated or unsaturated esters of the acids,preferably C.sub.10-C.sub.24, more preferably C.sub.16-C.sub_(—)24.Specific examples of the foregoing include oil soluble licorice extract,the glycyrrhizic and glycyrrhetic acids themselves, monoammoniumglycyrrhizinate, monopotassium glycyrrhizinate, dipotassiumglycyrrhizinate, 1-beta-glycyrrhetic acid, stearyl glycyrrhetinate, and3-stearyloxy-glycyrrhetinic acid, and disodium3-succinyloxy-beta-glycyrrhetinate. Stearyl glycyrrhetinate ispreferred.

Anti-Cellulite Agents

The compositions of the present invention may also contain a safe andeffective amount of an anti-cellulite, agent. Suitable agents mayinclude, but are not limited to, xanthine compounds (e.g., caffeine,theophylline, theobromine, and aminophylline).

Topical Anesthetics

The compositions of the present invention may also contain a safe andeffective amount of a topical anesthetic. Examples of topical anestheticdrugs include benzocaine, lidocaine, bupivacaine, chlorprocainc,dibucaine, etidocaine, mepivacaine, tetracaine, dyclonine, hexylcaine,procaine, cocaine, ketamine, pramoxine, phenol, and pharmaceuticallyacceptable salts thereof.

Tanning Agents

The compositions of the present invention may contain a tanning agent.When present, it is preferable that the compositions contain from about0.1% to about 20%, more preferably from about 2% to about 7%, and stillmore preferably from about 3% to about 6%, by weight of the composition,of dihydroxyacetone as an artificial tanning agent.

Skin Lightening Agents

The compositions of the present invention may contain a skin lighteningagent. When used, the compositions preferably contain from about 0.1% toabout 10%, more preferably from about 0.2% to about 5%, also preferablyfrom about 0.5% to about 2%, by weight of the composition, of a skinlightening agent. Suitable skin lightening agents include those known inthe art, including kojic acid, arbutin, ascorbic acid and derivativesthereof (e.g., magnesium ascorbyl phosphate or sodium ascorbylphosphate), and extracts (e.g., mulberry extract, placental extract).Skin lightening agents suitable for use herein also include thosedescribed in the PCT publication No. 95/34280, in the name ofHillebrand, corresponding to PCT Application No. U.S. 95/07432, filedJun. 12, 1995; and co-pending U.S. application Ser. No. 08/390,152 filedin the names of Kvalnes, Mitchell A. DeLong, Barton J. Bradbury, CurtisB. Motley, and John D. Carter, corresponding to PCT Publication No.95/23780, published Sep. 8, 1995.

Skin Soothing and Skin Healing Agents

The compositions of the present invention may comprise a skin soothingor skin healing agent. Skin soothing or skin healing agents suitable foruse herein include panthenoic acid derivatives (including panthenol,dexpanthenol, ethyl panthenol), aloe vera, allantoin, bisabolol, anddipotassium glycyrrhizinate. A safe and effective amount of a skinsoothing or skin healing agent may be added to the present composition,preferably, from about 0.1% to about 30%, more preferably from about0.5% to about 20%, still more preferably from about 0.5% to about 10%,by weight of the composition formed.

Antimicrobial and Antifungal Agents

The compositions of the present invention may contain an antimicrobialor antifungal agent. Such agents are capable of destroying microbes,preventing the development of microbes or preventing the pathogenicaction of microbes. A safe and effective amount of an antimicrobial orantifungal agent may be added to the present compositions, preferably,from about 0.001% to about 10%, more preferably from about 0.01% toabout 5%, and still more preferably from about 0.05% to about 2%.

Examples of antimicrobial and antifungal agents include B-lactam drugs,quinolone drugs, ciprofloxacin, norfloxacin, tetracycline, erythromycin,amikacin, 2,4,4′-trichloro-2′-hydroxy diphenyl ether,3,4,4′-trichlorobanilide, phenoxyethanol, phenoxy propanol,phenoxyisopropanol, doxycycline, capreomycin, chlorhexi dine,chlortetracycline, oxytetracycline, clindamycin, ethambutol, hexamidineisethionate, metronidazole, pentamidine, gentamicin, kanamycin,lineomycin, methacycline, methenamine, minocycline, neomycin,netilmicin, paromomycin, streptomycin, tobramycin, miconazole,tetracycline hydrochloride, erythromycin, zinc erythromycin,erythromycin estolate, erythromycin stearate, amikacin sulfate,doxycycline hydrochloride, capreomycin sulfate, chlorhexidine gluconate,chlorhexidine hydrochloride, chlortetracycline hydrochloride,oxytetracycline hydrochloride, clindamycin hydrochloride, ethambutolhydrochloride, metronidazole hydrochloride, pentamidine hydrochloride,gentamicin sulfate, kanamycin sulfate, lineomycin hydrochloride,methacycline hydrochloride, methenamine hippurate, methenaminemandelate, minocycline hydrochloride, neomycin sulfate, netilmicinsulfate, paromomycin sulfate, streptomycin sulfate, tobramycin sulfate,miconazole hydrochloride, ketaconazole, amanfadine hydrochloride,amanfadine sulfate, octopirox, parachlorometa xylenol, nystatin,tolnaftate, zinc pyrithione and clotrimazole.

Additionally antimicrobial peptides can be used.

Sunscreen Agents

Exposure to ultraviolet light can result in excessive scaling andtexture changes of the stratum corneum. Therefore, the compositions ofthe subject invention may optionally contain a sunscreen agent. As usedherein, “sunscreen agent” includes both sunscreen agents and physicalsunblocks. Suitable sunscreen agents may be organic or inorganic.

Inorganic sunscreens useful herein include the following metallicoxides; titanium dioxide having an average primary particle size of fromabout 15 nm to about 100 nm, zinc oxide having an average primaryparticle size of from about 15 nm to about 150 nm, zirconium oxidehaving an average primary particle size of from about 15 nm to about 150nm, iron oxide having an average primary particle size of from about 15nm to about 500 nm, and mixtures thereof. When used herein, theinorganic sunscreens are present in the amount of from about 0.1% toabout 20%, preferably from about 0.5% to about 10%, more preferably fromabout 1% to about 5%, by weight of the composition.

A wide variety of conventional organic sunscreen agents are suitable foruse herein. Sagarin, et al., at Chapter VIII, pages 189 et seq., ofCosmetics Science and Technology (1972), discloses numerous suitableagents. Specific suitable sunscreen agents include, for example:p-aminobenzoic acid, its salts and its derivatives (ethyl, isobutyl,glyceryl esters; p-dimethylaminobenzoic acid); anthranilates (i.e.,o-amino-benzoates; methyl, menthyl, phenyl, benzyl, phenylethyl,linalyl, terpinyl, and cyclohexenyl esters); salicylates (amyl, phenyl,octyl, benzyl, menthyl, glyceryl, and di-pro-pyleneglycol esters);cinnamic acid derivatives (menthyl and benzyl esters, a-phenylcinnamonitrile; butyl cinnamoyl pyruvate); dihydroxycinnamic acidderivatives (umbelliferone, methylumbelliferone,methylaceto-umbelliferone); trihydroxy-cinnamic acid derivatives(esculetin, methylesculetin, daphnetin, and the glucosides, esculin anddaphnin); hydrocarbons (diphenylbutadiene, stilbene); dibenzalacetoneand benzalacetophenone; naphtholsulfonates (sodium salts of2-naphthol-3,6-disulfonic and of 2-naphthol-6,8-disulfonic acids);di-hydroxynaphthoic acid and its salts; o- andp-hydroxybiphenyldisulfonates; coumarin derivatives (7-hydroxy,7-methyl, 3-phenyl); diazoles (2-acetyl-3-bromoindazole, phenylbenzoxazole, methyl naphthoxazole, various aryl benzothiazoles); quininesalts (bisulfate, sulfate, chloride, oleate, and tannate); quinolinederivatives (8-hydroxyquinoline salts, 2-phenylquinoline); hydroxy- ormethoxy-substituted benzophenones; uric and violuric acids; tannic acidand its derivatives (e.g., hexaethylether); (butyl carbotol) (6-propylpiperonyl)ether; hydroquinone; benzophenones (oxybenzene, sulisobenzone,dioxybenzone, benzoresorcinol, 2,2′,4,4′-tetrahydroxybenzophenone,2,2′-dihydroxy-4,4′-dimethoxybenzophenone, octabenzone;4-isopropyldibenzoylmethane; butylmethoxydibenzoylmethane; etocrylene;octocrylene; [3-(4′-methylbenzylidene bornan-2-one), terephthalylidenedicamphor sulfonic acid and 4-isopropyl-di-benzoylmethane.

Conditioning Agents

The compositions of the present invention may contain a conditioningagent selected from humectants, moisturizers, or skin conditioners. Avariety of these materials can be employed and each can be present at alevel of from about 0.01% to about 20%, more preferably from about 0.1%to about 10%, and still more preferably from about 0.5% to about 7% byweight of the composition. These materials include, but are not limitedto, guanidine; urea; glycolic acid and glycolate salts (e.g. ammoniumand quaternary alkyl ammonium); salicylic acid; lactic acid and lactatesalts (e.g., ammonium and quaternary alkyl ammonium); aloe vera in anyof its variety of forms (e.g., aloe vera gel); polyhydroxy alcohols suchas sorbitol, mannitol, xylitol, erythritol, glycerol, hexanetriol,butanetriol, propylene glycol, butylene glycol, hexylene glycol and thelike; polyethylene glycols; sugars (e.g., melibiose) and starches; sugarand starch derivatives (e.g., alkoxylated glucose, fucose, glucosamine);hyaluronic acid; lactamide monoethanolamine; acetamide monoethanolamine;panthenol; allantoin; and mixtures thereof. Also useful herein are thepropoxylated glycerols described in U.S. Pat. No. 4,976,953, to Orr etal, issued Dec. 11, 1990.

Structuring Agents

The compositions hereof, and especially the emulsions hereof, maycontain a structuring agent. Structuring agents are particularlypreferred in the oil-in-water emulsions of the present invention.Without being limited by theory, it is believed that the structuringagent assists in providing rheological characteristics to thecomposition which contribute to the stability of the composition. Forexample, the structuring agent tends to assist in the formation of theliquid crystalline gel network structures. The structuring agent mayalso function as an emulsifier or surfactant. Preferred compositions ofthis invention contain from about 0.1% to about 20%, more preferablyfrom about 0.1% to about 10%, still more preferably from about 0.5% toabout 9%, of one or more structuring agents.

The preferred structuring agents of the present invention are selectedfrom stearic acid, palmitic acid, stearyl alcohol, cetyl alcohol,behenyl alcohol, stearic acid, palmitic acid, the polyethylene glycolether of stearyl alcohol having an average of about 1 to about 5ethylene oxide units, the polyethylene glycol ether of cetyl alcoholhaving an average of about 1 to about 5 ethylene oxide units, andmixtures thereof. More preferred structuring agents of the presentinvention are selected from stearyl alcohol, cetyl alcohol, behenylalcohol, the polyethylene glycol ether of stearyl alcohol having anaverage of about 2 ethylene oxide units (steareth-2), the polyethyleneglycol ether of cetyl alcohol having an average of about 2 ethyleneoxide units, and mixtures thereof. Even more preferred structuringagents are selected from stearic acid, palmitic acid, stearyl alcohol,cetyl alcohol, behenyl alcohol, steareth-2, and mixtures thereof.

Thickening Agent (Including Thickeners and Gelling Agents)

The compositions of the present invention can contain one or morethickening agents, preferably from about 0.1% to about 5%, morepreferably from about 0.1% to about 4%, and still more preferably fromabout 0.25% to about 3%, by weight of the composition.

Nonlimiting classes of thickening agents for use in the compositions ofthe invention include those selected from the following: carboxylic acidpolymers (such as those described in U.S. Pat. No. 5,087,445, to Haffeyet al, issued Feb. 11, 1992; U.S. Pat. No. 4,509,949, to Huang et al,issued Apr. 5, 1985; U.S. Pat. No. 2,798,053, to Brown, issued Jul. 2,1957; and in CTFA International Cosmetic Ingredient Dictionary, FourthEdition, 1991, pp. 12 and 80); crosslinked polyacrylate polymers (suchas those described in U.S. Pat. No. 5,100,660, to Hawe et al, issuedMar. 31, 1992; U.S. Pat. No. 4,849,484, to Heard, issued Jul. 18, 1989;U.S. Pat. No. 4,835,206, to Farrar et al, issued May 30, 1989; U.S. Pat.No. 4,628,078 to Glover et al issued Dec. 9, 1986; U.S. Pat. No.4,599,379 to Flesher et al issued Jul. 8, 1986; and EP 228,868, toFarrar et al, published Jul. 15, 1987); polyacrylamide polymers (such asnonionic polyacrylamide polymers including substituted branched orunbranched polymers and multi-block copolymers of acrylamides andsubstituted acrylamides with acrylic acids and substituted acrylicacids); polysaccharides (which refers to gelling agents that contain abackbone of repeating sugar (i.e., carbohydrate) units, includingcellulose, carboxymethyl hydroxyethylcellulose, cellulose acetatepropionate carboxylate, hydroxyethylcellulose, hydroxyethylethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose,methyl hydroxyethylcellulose, microcrystalline cellulose, sodiumcellulose sulfate, and mixtures thereof); and gums (such as acacia,agar, algin, alginic acid, ammonium alginate, amylopectin, calciumalginate, calcium carrageenan, carnitine, carrageenan, dextrin, gelatin,gellan gum, guar gum, guar hydroxypropyltrimonium chloride, hectorite,hyaluroinic acid, hydrated silica, hydroxypropyl chitosan, hydroxypropylguar, karaya gum, kelp, locust bean gum, natto gum, potassium alginate,potassium carrageenan, propylene glycol alginate, sclerotium gum, sodiumcarboyxmethyl dextran, sodium carrageenan, tragacanth gum, xanthan gum,and mixtures thereof).

Composition Preparation

The compositions useful for the methods of the present invention aregenerally prepared by conventional methods such as are known in the artof making topical compositions. Such methods typically involve mixing ofthe ingredients in one or more steps to a relatively uniform state, withor without heating, cooling, application of vacuum, and the like.

Preservatives

Preservatives can be incorporated into the compositions of the presentinvention to protect against the growth of potentially harmfulmicroorganisms. While it is in the aqueous phase that microorganismstend to grow, microorganisms can also reside in the anhydrous or oilphase. As such, preservatives, which have solubility in both water andoil, are preferably employed in the present compositions. Suitabletraditional preservatives for compositions of this invention are alkylesters of parahydroxybenzoic acid. Other preservatives, which can beused include hydantoin derivatives, propionate salts, and a variety ofquaternary ammonium compounds.

Particularly preferred preservatives are methylparaben, imidazolidinylurea, sodium dehydroacetate, propylparaben, trisodium ethylenediaminetetraacetate (EDTA), and benzyl alcohol. The preservative can beselected to avoid possible incompatibilities between the preservativeand other ingredients. Preservatives are preferably employed in amountsranging from about 0.01% to about 2% by weight of the composition. Otherpreservatives known in the art can be used in the present invention.

Methods of Administration

Another aspect of the invention is to provide a method of administeringa composition of the invention, wherein dispersed lipid vesicles and/orcyclodextrins comprising collagen are provided to the dermal layer of apatient's skin. The method includes the step of contacting the skin orother target site of the subject with a composition including a lipidvesicle (e.g., non-phospholipid paucilamellar lipid vesicle) having acavity containing human collagen.

The compositions of the present invention are useful for regulatingand/or improving mammalian skin condition. Such regulation of epidermaltissue conditions can include prophylactic and therapeutic regulation.For example, such regulating methods are directed to thickening dermaltissue and preventing and/or retarding atrophy of mammalian skin,preventing and/or retarding the appearance of spider vessels and/or redblotchiness on mammalian skin, preventing and/or retarding theappearance of dark circles under the eye of a mammal, preventing and/orretarding sallowness of mammalian skin, preventing and/or retardingsagging of mammalian skin, softening and/or smoothing lips of a mammal,preventing and/or relieving itch of mammalian skin, regulating skintexture (e.g. wrinkles and fine lines), and improving skin color (e.g.redness, freckles).

Regulating epidermal tissue condition involves topically applying to theepidermal tissue a safe and effective amount of a composition of thepresent invention. The amount of the composition which is applied, thefrequency of application and the period of use will vary widelydepending upon the level of collagen (and, when present, other skin careagents) of a given composition and the level of regulation desired,e.g., in light of the level of epidermal tissue damage present orexpected to occur.

In a preferred embodiment, the composition is chronically applied to theskin. By “chronic topical application” is meant continued topicalapplication of the composition over an extended period during thesubject's lifetime, preferably for a period of at least about one week,more preferably for a period of at least about one month, even morepreferably for at least about three months, even more preferably for atleast about six months, and more preferably still for at least about oneyear. While benefits are obtainable after various maximum periods of use(e.g., five, ten or twenty years), it is preferred that chronicapplication continue throughout the subject's lifetime. Typicallyapplications would be on the order of about once per day over suchextended periods, however application rates can vary from about once perweek up to about three times per day or more.

A wide range of quantities of the compositions of the present inventioncan be employed to provide a skin appearance and/or feel benefit.Quantities of the present compositions which are typically applied perapplication are, in mg composition/cm² skin, from about 0.1 mg/cm² toabout 10 mg/cm². A particularly useful application amount is about 1mg/cm² to about 2 mg/cm².

Improving and/or regulating epidermal tissue condition is preferablypracticed by applying a composition in the form of a skin lotion, cream,gel, foam, ointment, paste, emulsion, spray, conditioner, tonic,cosmetic, lipstick, foundation, after-shave, or the like which ispreferably intended to be left on the skin or other keratin structurefor some esthetic, prophylactic, therapeutic or other benefit (i.e., a“leave-on” composition). After applying the composition to the skin, itis preferably left on the skin for a period of at least about 15minutes, more preferably at least about 30 minutes, even more preferablyat least about 1 hour, still more preferably for at least several hours,e.g., up to about 12 hours. Any part of the external portion of the bodycan be treated, e.g., lips, under-eye area, eyelids, scalp, neck, torso,arms, hands, legs, feet, etc. The composition can be applied with thefingers or with an implement or device (e.g., pad, cotton ball,applicator pen, spray applicator, and the like).

Another approach to ensure a continuous dispersal of at least a minimumlevel of collagen (and, when present, at least one skin care agent) tothe dermal layer is to apply the compound by use of a patch applied,e.g., to the face. Such an approach is particularly useful for problemskin areas needing more intensive treatment (e.g., facial crows feetarea, frown lines, under eye area, and the like). The patch can beocclusive, semi-occlusive or non-occlusive and can be adhesive ornon-adhesive. The composition can be contained within the patch or beapplied to the skin prior to application of the patch. The patch canalso include additional agents such as chemical initiators forexothermic reactions such as those described in U.S. Pat. Nos.5,821,250, 5,981,547, and 5,972,957 to Wu, et al. The patch ispreferably left on the skin for a period of at least about 5 minutes,more preferably at least about 15 minutes, more preferably still atleast about 30 minutes, even more preferably at least about 1 hour,still more preferably at night as a form of night therapy.

Example 1 Absorption of Collagen into Skin

The study evaluated the percutaneous absorption pharmacokinetics of¹⁴C-Collagen spiked to a liposome. Absorption was measured in excisedhuman face skin, in vitro, using the finite dose technique and FranzDiffusion Cells.

The in vitro human cadaver skin model has proven to be a valuable toolfor the study of percutaneous absorption and the determination of thepharmacokinetics of topically applied drugs. The model uses humancadaver skin mounted in specially designed diffusion cells that allowthe skin to be maintained at a temperature and humidity that matchtypical in vivo conditions. A finite dose (e.g. 4-7 mg/cm²) offormulation is applied to the outer surface of the skin and drugabsorption is measured by monitoring its rate of appearance in thereceptor solution bathing the inner surface of the skin.

Test Article - Day Cream Tracer - ¹⁴C Collagen (ARC 2005) (methyl-14C)

The product was tested on replicate skin sections from three differentskin donors, for the percutaneous absorption of ¹⁴C-Collagen spikedformulation over a 48-hour dose period. At pre-selected times afterdosing, the dermal reservoir solution was removed in its entirety,replaced with fresh receptor solution, and an aliquot saved forsubsequent analysis. At the completion of the study, the dermal andepidermal contents were also evaluated. The samples were analyzed for¹⁴C isotope content by liquid scintillation spectroscopy.

SUMMARY TABLE [¹⁴C]-Collagen spiked Novasome Total Absorption ResultsAcross Donor Skin Source Total Pen* (μg) Total Pen (%) Eyelid 3.83 ±2.40  42.96 ± 21.21 Pre-Auricular 2.63 ± 0.56 19.63 ± 1.27 Combined Data3.03 ± 0.51 27.40 ± 7.81 Percutaneous Absorption of ¹⁴C-Collagen asradioisotope through Excised Human Face Skin over 48 hours from a SingleApplication. Mean ± SE (n = 3) as Total Mass (μg) and Percent of AppliedDose *“Pen” is penetration

The radiolabeled collagen was prepared by American RadiolabeledChemicals, Inc. (ARC; St. Louis, Mo. 63143). Briefly, the study Sponsorsent their collagen to ARC for labeling. ARC labeled the collagen bymethylation using [¹⁴C] formaldehyde and sodium cyanoborohydride. Theradiolabeled material was indicated to have a specific activity of 25μCi/mg and was provided in a 1 mL volume of 0.01M potassium phosphatebuffer (pH 7.2).

The radiolabeled material was first dried by vacuum centrifugation(Speed Vac, Savant, Inc.) for approximately 3 hours, followed byreconstitution in 50 μL of the Novasome Day Cream and mixed by dualsyringe (100 μL) cross extrusion through a 3-way stop-cock valve 20times. The spiked cream was allowed to equilibrate for 24 hrs at roomtemperature prior to use. Final specific activity was measured as 0.1μCi/μL cream formulation with 3.82 μg/uL radiolabeled collagencontributing to the cream base.

Study Skin Preparation:

Human excised face skin (eyelid and pre-auricular) without obvious signsof skin disease, obtained following cosmetic surgery, was used in thisstudy. It was cleared of all subcutaneous tissue and the lower ˜25% ofthe dermis, sealed in water-impermeable plastic bags and stored at ≦−70°C. until used. Prior to the experiment, skin was thawed, and then rinsedin water to remove any adherent blood or other material from thesurface.

Skin from a single donor was cut into multiple smaller sections largeenough to fit on Franz diffusion cells (0.4 cm²-0.8 cm²). The dermalchamber was filled to capacity with a reservoir solution ofphosphate-buffered isotonic saline (PBS), pH 7.4±0.1, and the epidermalchamber was left open to the ambient laboratory environment. The cellswere then placed in a diffusion apparatus in which the dermal reservoirsolution was stirred magnetically at approximately 600 RPM and its skinsurface temperature maintained at 32.0°±1.0° C.

To assure the integrity of each skin section, its permeability totritiated water was determined before application of the test products.Following a brief (0.5-1 hour) equilibrium period, ³H₂O (NEN, Boston,Mass., sp. Act. ˜0.5 μCi/mL) was layered across the top of the skin bydropper so that the entire exposed surface was covered (approximately100-500 μL). After 5 minutes the ³H₂O aqueous layer was removed. At 30minutes the receptor solution was collected and analyzed for radioactivecontent by liquid scintillation counting. Following the integrity testthe receptor solution was changed multiple times to remove anymeasurable residual tritium.

Dosing and Sample Collection

Prior to administration of the topical test formulations to the skinsections, the chimney was removed from the Franz Cell to allow fullaccess to the epidermal surface of the skin and the reservoir solutionwas replaced with afresh solution of PBS.

Subsequently, the test product was applied to replicate sections of thesame donor skin. Dosing was performed using a positive displacementpipette set to deliver 5 μL formulations/cm². Given the available skin,three chamber sizes with different dosing area were using (0.4, 0.5 and0.8 cm²). The dose was spread throughout the surface with the tip of thepipette. Five to ten minutes after application the chimney portion ofthe Franz Cell was replaced. Spare cells were not dosed, but sampled, toevaluate for interfering substances during the analytical analysis.

At pre-selected time intervals after test formulation application (0.5,1, 3, 7, 19, 26, 43, 48 hr) the receptor solution was removed in itsentirety replaced with fresh solution, and an aliquot taken foranalysis.

After the last sample was collected, the surface of the skin was washedwith double distilled de-ionized water to collect un-absorbedformulation form the surface of the skin. Following the wash, the skinwas removed from the chamber, separated into epidermis and dermis, andeach processed for subsequent analysis for isotope content.

Analytical Methods

Analysis for ¹⁴C-isotope content of each sample was by liquidscintillation spectroscopy. One-milliliter volumes of each receptorsolution and each surface wash solution received 5-7 mL scintillationfluid. Tissue (epidermis and dermis) was dissolved in Soluene-350(PerkinElmer; Lot# 24-060203) overnight, following which each samplereceived 5-7 mL of scintillation fluid.

Samples were quantified for ¹⁴C content by liquid scintillationspectroscopy using a PerkinElmer Tri-Carb 3100TR liquid scintillationcounter. Each sample was counted for no less than 5 minutes each, induplicate. Counts per minute (CPM) were automatically converted todecays per minute (DPM) using the external standard quench correctionmethod. All data were corrected for isotope background from blanksamples.

TABLE 1 Rate of Penetration as Mean Flux (μg/cm²/hr) Results Time (hr)*Eyelid Pre-Auricular Combined Data 0.25 0.14 ± 0.10 0.04 ± 0.02 0.07 ±0.04 0.75 0.20 ± 0.02 0.12 ± 0.00 0.15 ± 0.03 2.0 0.18 ± 0.01 0.15 ±0.06 0.16 ± 0.03 5.0 0.21 ± 0.09 0.10 ± 0.04 0.14 ± 0.04 13.0 0.17 ±0.07 0.07 ± 0.01 0.11 ± 0.03 22.5 0.23 ± 0.18 0.08 ± 0.00 0.13 ± 0.0535.0 0.10 ± 0.01 0.07 ± 0.00 0.08 ± 0.01 45.5 0.30 ± 0.30 0.07 ± 0.010.15 ± 0.08 Percutaneous Absorption of ¹⁴C-Collagen as radioisotopethrough Human Cadaver Skin over 48 hours from a Single Application (Mean± SD Eyelid, Mean ± SE Pre-Auricular, n = 1-2 Donors). *Time as midpointbetween samples.

TABLE 2 Total Absorption and Mass Balance Results Parameter EyelidPre-Auricular Combined Data Total Absorption 3.83 ± 2.40 2.63 ± 0.563.03 ± 0.51 (μg) Dermis (μg) 0.02 ± 0.01 0.03 ± 0.01 0.02 ± 0.01Epidermis (μg) 0.13 ± 0.06 0.28 ± 0.13 0.23 ± 0.09 Surface Wash (μg)5.37 ± 0.25 9.73 ± 1.65 8.28 ± 1.74 Total Absorption (5) 42.96 ± 21.2119.63 ± 1.27  27.40 ± 7.81  Dermis (%) 0.20 ± 0.11 0.18 ± 0.03 0.19 ±0.02 Epidermis (%) 1.51 ± 0.48 2.01 ± 0.66 1.84 ± 0.42 Surface Wash (%)63.14 ± 7.04  74.10 ± 0.48  70.45 ± 3.67  Total Recovery (%) 107.81 ±14.76  95.92 ± 2.44  99.88 ± 4.21  Percutaneous Absorption of¹⁴C-Collagen as radioisotope through Human Cadaver Skin over 48 hoursfrom a Single Application. Mean ± SD Eyelid, and Mean ± SE Pre-Auricularas Percent of Applied Dose and Total Mass (μg). (n = 1-2 Donors)

The data indicated that when ¹⁴C-Collagen is incorporated into theNovasome Day Cream base formulation, radioisotope penetrates into andthrough human excised face skin.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A liposome comprising at least one component selected from the groupconsisting of hyaluronic acid and collagen.
 2. The liposome, accordingto claim 1, comprising hyaluronic acid.
 3. The liposome, according toclaim 2, wherein the hyaluronic acid is cross-linked.
 4. The liposome,according to claim 2, wherein the hyaluronic acid is not cross-linked.5. The liposome, according to claim 1, wherein the hyaluronic acid islinear hyaluronic acid of less than 500 kDa.
 6. The liposome, accordingto claim 1, comprising collagen.
 7. The liposome, according to claim 6,wherein the collagen is human collagen.
 8. The liposome, according toclaim 6, wherein the collagen is a recombinant collagen.
 9. Theliposome, according to claim 6, wherein said collagen is a Type I orType III collagen.
 10. The liposome, according to claim 6, wherein saidcollagen is in a particle size of less than 800 nm.
 11. The liposome,according to claim 6, wherein said collagen is in the fibril form. 12.The liposome, according to claim 1, which comprises collagen andhyaluronic acid.
 13. The liposome, according to claim 1, furthercomprising at least one of the group consisting of desquamation agents,anti-acne agents, anti-wrinkle agents, vitamin. B₃ compounds, retinoids,hydroxyl acids, anti-oxidants, radical scavengers, chelators,flavonoids, anti-inflammatory agents, anti-cellulite agents, topicalanesthetics, tanning agents, skin lightening agents, skin soothing andskin healing agents, antimicrobial and antifungal agents, sunscreenagents, conditioning agents, structuring agents, thickening agent, andpreservatives.
 14. The liposome, according to claim 1, which ispaucilamellar.
 15. The liposome, according to claim 1, which comprisesnon-phospholipid surfactants.
 16. A composition for delivering an agentinto the skin of a subject wherein said composition comprises acyclodextrin having associated therewith the agent, wherein the agent isselected from the group consisting of collagen and hyaluronic acid. 17.A method for administering an agent into the skin of a subject, themethod comprising the step of contacting the skin of the subject with acomposition comprising a carrier comprising a cavity having encapsulatedtherein an agent selected from the group consisting of collagen andhyaluronic acid, wherein said carrier is a liposome or a cyclodextrin.18. The method, according to claim 17, wherein said carrier is aliposome.
 19. The method, according to claim 18, wherein the liposomecomprises hyaluronic acid.
 20. The method, according to claim 18,wherein the liposome is paucilamellar.